Synchrotron X-ray microtomography (on a micron scale) provides three-dimensional imaging representation of bone ingrowth in calcium phosphate biomaterials.

This study used synchrotron X-ray microtomography on a micron scale to compare three-dimensional (3D) bone ingrowth after implantation of various calcium phosphate bone substitutes in a rabbit model. The advantage of using this new method for the study of biomaterials was then compared with histomorphometry for analysis of interconnection and bone ingrowth. The study focused on the newly formed bone-biomaterial interface. Macroporous Biphasic Calcium Phosphate (MBCP) ceramic blocks and two different injectable calcium phosphate biomaterials [an injectable bone substitute (IBS) consisting of a biphasic calcium phosphate granule suspension in hydrosoluble polymer and a calcium phosphate cement material (CPC)] were studied after in vivo implantation. Absorption or phase-contrast microtomography was performed with the dedicated set-up at beamline ID22. Experimental spatial resolution was between 1 and 1.4 microm, depending on experimental radiation. All calcium phosphates tested showed osteoconduction. IBS observations after 3D reconstruction showed interconnected bioactive biomaterial with total open macroporosity and complete bone ingrowth as early as 3 weeks after implantation. This experimentation was consistent with two-dimensional histomorphometric analysis, which confirmed its suitability for biomaterials. This 3D study relates the different types of bone substitution to biomaterial architecture. As porosity and interconnection increase, bone ingrowth becomes greater at the expense of the bone substitute: IBS>MBCP>CPC.

Noninvasive bone replacement with a new injectable calcium phosphate biomaterial.

The use of injectable calcium phosphate (CaP) biomaterials in noninvasive surgery should provide efficient bone colonization and implantation. Two different kinds of injectable biomaterials are presently under development: ionic hydraulic bone cements that harden in vivo after injection, and an association of biphasic calcium phosphate (BCP) ceramic granules and a water-soluble polymer vehicle (a technique particularly investigated by our group), providing an injectable CaP bone substitute (IBS). In our study, we compared these two approaches, using physicochemical characterizations and in vivo evaluations in light microscopy, scanning electron microscopy, and three-dimensional microtomography with synchrotron technology. Three weeks after implantation in rabbit bone, both biomaterials showed perfect biocompatibility and bioactivity, but new bone formation and degradation of the biomaterial were significantly greater for BCP granules than for ionic cement. Newly formed bone developed, binding the BCP granules together, whereas new bone grew only on the surface of the cement, which remained dense, with no obvious degradation 3 weeks after implantation. This study confirms that BCP granules carried by a cellulosic polymer conserve bioactivity and are conducive to earlier and more extensive bone substitution than a carbonated-hydroxyapatite bone cement. The presence of intergranular spaces in the BCP preparation, as shown on microtomography imaging, seems particularly favorable, allowing body fluids to reach each BCP granule immediately after implantation. Thus, the IBS functions as a completely interconnected ceramic with total open macroporosity. This new bone replacement approach should facilitate microinvasive bone surgery and local delivery of bone therapy agents.

Crystallization at the polymer/calcium-phosphate interface in a sterilized injectable bone substitute IBS.

Calcium phosphate (CaP) ceramics are the main raw materials used to elaborate blocks or granules for bone substitutes. In this study, injectable bone substitutes (IBS) were developed for applications in orthopedic or dental surgery. Sterile, ready-to-use composite containing CaP granules (biphasic calcium phosphate, BCP) and polymer (hydroxypropylmethylcellulose, HPMC) was prepared. Steam sterilization produced new phenomena at the CaP/polymer interface, resulting in crystal growth. These phenomena may constitute a model for the biomineralization study. Scanning electron microscopy showed that the formed crystallites organize themselves into a three-dimensional structure. Currently, the mechanisms of crystal growth are unknown and have been observed with only one combination of polymer/BCP ceramics after steam sterilization.

Skin sensitization study of two hydroxypropyl methylcellulose components (Benecel and E4M) of an injectable bone substitute in guinea pigs.

Although initial results were promising for an injectable bone substitute (IBS) associating a hydroxypropyl methylcellulose (HPMC) polymer vector (Benecel, 2 w/w %) with biphasic calcium phosphate (BCP), a sensitization reaction occurred probably related to the degree of polymer purity. In this context, Benecel and another HPMC, E4M were investigated in the present study. The expected composition of the polymers was confirmed by gas-liquid chromatography. Studies in the guinea pig showed that Benecel has strong sensitization capacity and E4M none. Benecel manifests impurities (30 times more than E4M) in individual fibers or rounded clumps that are apparently responsible for extreme sensitization. Purification by ultracentrifugation associated with 0.2 microm filtration can decrease sensitization capacity considerably, though with a slight loss of polymer concentration. Fourier transform infrared (FTIR) analysis showed that the impurities were largely cellulose derivatives. However, extraction by organic solvent, followed by FTIR studies and micro-X analysis, detected an oily substance containing carbon and silicon associated with the cellulose derivatives. E4M, a polymer with no sensitization capacity, could replace Benecel and improve results with IBS.

Interaction between hydroxypropyl methylcellulose and biphasic calcium phosphate after steam sterilisation: capillary gas chromatography studies.

The purpose of this study was to check the chemical stability of an injectable bone substitute (IBS) composed of a 50/50 w/w mixture of 2.92% hydroxypropyl methylcellulose (HPMC) solution in deionized water containing biphasic calcium phosphate (BCP) granules (60% hydroxyapatite/40% beta-tricalcium phosphate w/w). After separation of the organic and mineral phases, capillary gas chromatography (GC) was used to study the possible modification of HPMC due to the contact with BCP granules following steam sterilisation and 32 days storage at room temperature. HPMC was extracted from IBS in aqueous medium, and a dialytic method was then used to extract calcium phosphate salts from the HPMC. The percentage of HPMC extracted from BCP was 98.5%+/-0.5%, as measured by UV. GC showed no chemical modifications after steam sterilisation and storage.