Bone response and mechanical strength of rabbit femoral defects filled with injectable CaP cements containing TGF-β1 loaded gelatin microparticles
Introduction
Calcium phosphate (CaP) ceramics are widely used as bone substitutes in reconstructive orthopedic and oral surgery because of their beneficial effects on bone healing. These ceramics can be applied as granules or prefabricated porous blocks, but they can also be formulated as an injectable CaP paste that can be shaped according to the required dimensions [1], [2], [3], [4], [5]. These so-called CaP cements are highly compatible with soft and hard tissues after setting in situ [6]. In view of tissue reconstruction, CaP cements are supposed to be subject of biological degradation and concomitant replacement by bone tissue. However, the degradation of CaP cements is known to be slow [7], which is likely due to the limited extent of porosity. CaP cements contain only an intrinsic nanoporosity that allows transport of nutrients and waste through the material, but the dimensions of this nanoporosity are insufficient to obtain tissue ingrowth [6]. As a consequence, attempts have been made to increase the porosity of the material to allow tissue ingrowth and to accelerate degradation [8], [9]. For the additional creation of microporosity, different methods have already been applied; the most commonly used method at our department is the incorporation of high molecular weight poly(lactic-co-glycolic acid) (HMW-PLGA) microparticles to generate CaP/PLGA composites [9], [10], [11], [12]. A limitation regarding the use of PLGA for microparticle production is the relative slow degradation of this polymer (6–12 weeks) [11]. This slow degradation still prevents cells to penetrate into the implants early after in vivo application, which is the probable cause of delayed bone formation in the composites [9], [12], [13], [14].
Therefore, a method was developed to prepare composites in which microparticles of gelatin were incorporated into CaP cement [15], [16]. Gelatin microparticles are biodegradable, biocompatible, and non-toxic and can be crosslinked with glutaraldehyde in order to increase thermal and mechanical stability of the microparticles under physiological conditions [17]. Furthermore, the crosslinking agent concentration and reaction period can be varied, allowing the creation of microparticles with different degradation properties [17], [18], [19].
Inherent to the creation of porosity is the loss of mechanical strength of the CaP cement material. This makes the composites less suitable for use under load-bearing conditions. In an ideal situation, the cement would retain its mechanical strength, while being resorbed and replaced by newly formed bone. Previous research already showed that the decreased mechanical properties are partly compensated by the ingrowth of bone in the cement porosity [12].
To further stimulate bone formation and cement resorption, microparticles can be used for the delivery of an appropriate growth factor. Among the candidate growth factors for such an application is transforming growth factor β1 (TGF-β1), which plays a significant role in wound healing [20], [21], [22] by enhancing the repair of injured tissue like skin and bone [23]. TGF-β1 acts on osteoblasts, chondrocytes, and cells of the osteoclastic lineage [24] and has been reported to stimulate osteogenesis at orthotopic sites [25], [26], [27].
In view of the above mentioned, this study investigated the potential of TGF-β1 loaded gelatin microparticles to enhance the bone response and mechanical strength of rabbit femoral defects filled with injectable CaP/gelatin microparticle composites.
Section snippets
Calcium phosphate (CaP) cement
CaP cement (Calcibon®; Merck Biomaterial GmbH, Darmstadt, Germany) was used for the preparation of the implants. The chemical composition of this cement is 61% α tri-calcium phosphate (α-TCP), 26% CaHPO4, 10% CaCO3 and 3% precipitated hydroxyapatite (PHA). Before usage, the cement powder was sterilized by gamma radiation with 25 kGy (Isotron B.V., Ede, The Netherlands).
Gelatin microparticles
Gelatin microparticles were prepared as described by Holland et al. [28]. Briefly, a gelatin solution was prepared by dissolving
Composite characterization
The distribution of the unswollen gelatin microparticles varied between 1 and 49 μm with an average size of 8.4±7.6 μm, while the water-swollen microparticles varied between 1 and 66 μm with an average size of 20.7±14.6 μm. The porosity of the composite formulations after setting was 45.0±1.3% (Table 1).
General observations of animals
All 36 rabbits in this experiment remained in good health and did not show any wound complications after surgery. The original defects were completely filled with the injectable composites without
Discussion
In this study, the bone response and mechanical strength of a rabbit femoral defect filled with an injectable CaP cement containing gelatin microparticles, either or not loaded with TGF-β1, was examined. The loading of gelatin microparticles with TGF-β1 was enrolled as a parameter to evaluate potential effects of this growth factor on bone formation and cement resorption.
The porosity of both composite formulations (CaP/gelatin with or without TGF-β1) was 45.0±1.3%, which should be high enough
Conclusions
In conclusion, this study demonstrated that after injection and setting of a CaP/gelatin composite, the mechanical strength of the composite including the new bone formation, is sufficient after 12 weeks of implantation for load-bearing purposes. Further, degradation of the composites was observed, indicated by the loss of integrity of the composites, and by the decline in composite diameter and concomitant replacement by newly formed bone with increasing implantation time. The addition of TGF-β
Acknowledgments
The authors would like to thank the Dutch Technology Foundation (STW) applied science division of NWO and the technology program of the Ministry of Economic Affairs for their financial support in this Project (NGT6205).
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