Scanning and Transmission Electron Microscopy, and Electron Probe Analysis of the Interface Between Implants and Host Bone

Bioinert materials (e.g., alumina implants) and bioactive ceramics (e.g., calcium phosphate ceramics, glass -ceramics) are now extensively used in dentistry. However, the physico-chemical interactions at the interfaces between the implant and the host bone are poorly understood. The purpose of this study was to define the interactions at these interfaces using a combination of analytical techniques: light microscopy, scanning and transmission electron microscopy, electron probe microanalysis, X-ray microradiography, X-ray diffraction, and infrared specstroscopy. Bioinert (pure titanium) and bioactive materials (hydroxyapatite, beta-tricalcium phosphate and biphasic calcium phosphate) were implanted in dogs, and the implants, recovered after various periods of implantation, were analyzed. The results demonstrated the following: the bioactive materials interact with the biological fluid and the living tissues in a specific manner. This process includes biodissolution/biodegradation, apatite crystal precipitation, and bone formation on the implant surface at the expense of the material. The results are discussed according to the limitations of the analytical techniques used. The medical and chemical word COALESCENCE is suggested to describe the specific interactions of bioactive materials and INTERACTION for the phenomenon of physical contact of the bioinert materials with the host bone

Pulpal Response to Calcium Phosphate Materials. In Vivo Study of Calcium Phosphate Materials in Endodontics

The aim of this study was to determine if calcium phosphate (CaP) materials could be used to substitute for calcium hydroxide (CH) as a pulp capping agent. Especially prepared and characterized CaP materials with CH as the reference or control material were used for pulpcapping teeth of pigs, rats, and dogs. The CaP materials included: DCPD (dicalcium phosphate dihydrate), OCP (octacalcium phosphate), ß-TCP ({3-tricalcium phosphate), BCP (biphasic calcium phosphate mixture of 50150 HA and ß-TCP), and HA (hydroxyapatite) which were used in particle sizes of \u3c 5 μm or \u3c 150 μm. The animals were sacrificed after 21 days to 4 months after pulp-capping. The extracted teeth were immediately prepared for the following analyses: light microscopy, scanning electron microscopy (SEM) using backscattered electrons (BSE) , and energy dispersive Xray (EDX) microanalysis. Three types of mineralizations were observed: dentin bridge formation, dystrophic calcification and mineralization. All the CaP materials showed biocompatibility. Based on these results, it is suggested that the CaP materials tested may be useful for specific clinical applications in endodontics, e.g., pulp capping (microparticles of HA, TCP, BCP), and pulpectomy (HA, OCP, DCPD)

Heating of Calcium Phosphate Crystals: Morphological Consequences and Biological Implications

Sintering hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) affects the chemical composition, the crystallinity, and the morphological features as demonstrated by means of X-ray diffraction (XRD) , infrared spectroscopy (IR), and scanning electron microscopy (SEM). When heated to 1230°C, 16.7% of HA had decomposed to ß-TCP. SEM investigations showed homogeneous, sharp angular polyhedric blocks of 30 to 50 µm with rare surface pores. On heating at 1230°C, ß-TCP had entirely transformed to a-TCP. During sintering, the size of the powder grains increased and progressive bridging between the grains was observed. At 1230°C, a network within round-shaped polyhedric blocks of 50 to 90 µm was formed. In both, HA and IJ-TCP, surfaces were smooth. The chemical composition and the crystallinity of calcium phosphate ceramics determine their dissolution behavior and osteogenic properties. Nevertheless, their temperature dependent morphological features, such as, particle shape and size, surface texture, and porosity, as demonstrated in the present study, also influence the resorption rates , tissue responses, and wound healing duration. This should be emphasized more by clinicians in choosing an appropriate material for bone substitution

Osteoconductive properties of poly(96L/4D-lactide)/beta-tricalcium phosphate in long term animal model

The objective of this study was to determine the effect of calcium phosphate mineral content on the bone in-growth at the expense of composite of co-polylactide polymer charged with 2 different ratios of β-TCP granules (10 and 24 w-% of β-TCP). The evaluation was realized in a long term rabbit bone model. After 24, 48 and 76 weeks, the implants were examined by micro CT, scanning electron microscopy (SEM) using backscattered electron (BSE) and light microscopy (polarized and blue light microscopy). No foreign body reaction was detected during the 76 weeks follow-up in any of the test samples. Polymer hydrolysis began at approximately 24 weeks, by 76 weeks, the pure polymer implant had begun to release P(96L/4D)LA particles and show signs of peripheral localized bone resorption. A decrease in the amount of CaP was noticed between 24 and 76 weeks in both 10 wt-% and 24 wt-% β-TCP/P(96L/4D)LA composites. The study showed that the highest bone in-growth was with 24 wt-% β-TCP/P(96L/4D)LA composite. Bone in-growth and mineralization were evident for the composites associated with specific peripheral bone architecture. Fluorescent labelling demonstrated high bone in-growth and remodeling at the interface, while for pure co-polymer no bone remodeling or bone activity was maintained after 48 weeks. The study demonstrated the positive effect of calcium phosphate content into P(96L/4D)LA. This kind of composite is a suitable resorbable osteoconductive matrix, which provides long term stability required for ligament fixation device

Solution-Mediated Transformation of Octacalcium Phosphate (OCP) to Apatite

OCP crystals were hydrolyzed in solutions containing Ca2+, Mg2+, HPO42-, CO32-, F-, citrate or P2O7 ions. Products of hydrolysis were analyzed using scanning (SEM) and transmission (TEM) electron microscopy, infrared spectroscopy and x-ray diffraction. Results demonstrated that the OCP to Apatite (AP) transformation is influenced by: (1) types of ions in solution: inhibited by Mg2+, citrate or P2O74-; facilitated by F-, CO32-, HPO42- or Ca2+ ions; (2) ionic concentrations; (3) solution pH; (4) OCP crystal size. SEM showed needle-like micro-crystals on the surfaces and ends of OCP macrocrystals. TEM showed side-to-side and end-to-end arrangements and presence of central defects in the apatite crystals. IR spectra showed the incorporation of CO3, or HPO4, the HPO4 incorporation being least from F-containing solutions. These results suggest that OCP to AP transformation occurred by the process of dissolution of OCP and subsequent precipitation of Ca-deficient apatites, incorporating CO32-, HPO42- or F- present in solution. These results indicate that the observed stabilty of OCP in pathological calcifications may be due to the presence of Mg2+, citrate and/or P2O74- and/or low levels of CO32-, F-, Ca2+, HPO42- ions in the biological fluids

Biodegradation of Synthetic Biphasic Calcium Phosphate and Biological Calcified Substratum by Cells of Hemopoietic Origin

Different types of osteoclastic cells (authentic osteoclasit from human giant cell tumor and bone marrow of newborn rats; newly-formed osteoclasts from adult rat bone marrow), giant multinucleated cells and macrophages were studied for their effect on synthetic and natural mineralized substrata. Biphasic calcium phosphate ceramic consisted of hydroxyapatite and beta tricalcium phosphate was chosen for in vitro experiments, and dentine served as a positive control for cell resorbing activity . Our results show the limited capacity of authentic and newly-formed osteoclasts to resorb synthetic ceramic as compared to that of natural substrata. In vitro cell-mediated biodegradation included also modifications of the synthetic substratum surface caused presumably by phagocytosis of the material

Preliminary In Vivo Studies of a New Injectable Bone Substitute

Calcium-phosphate materials have been increasingly employed in orthopedic and dental applications in recent years and are now being developed for use in noninvasive surgery or as carriers for drug delivery systems. We developed an injectable bone substitute (IBS) for percutaneous orthopedic surgery which uses a biphasic calcium-phosphate (BCP) mixture composed of hydroxyapatite (60%) and ß-tricalcium phosphate (40%), together with a polymer (hydroxy-propyl-methyl-cellulose, HPMC) as a carrier. The best BCP/polymer ratio was determined to achieve the highest mineral phase in the composite and provide the rheological properties required for injectable material. The in vivo biocompatibility and biofunctionality of IBS were tested in rabbits using implants in subcutaneous, intramuscular and cartilage sites and defects created in trabecular bone of the femur epiphysis. The defects were filled with IBS, and histological studies were performed after 1, 2, 4 and 12 weeks on decalcified and non-decalcified sections. Image analysis and backscattered electron analyses were performed by scanning electron microscopy. This study demonstrated that HPMC is a non-toxic material which can be associated with calcium-phosphates to produce an IBS and create a matrix for deep cell colonization conducive to bone ingrowth

Reconstruction of irradiated bone segmental defects with a biomaterial associating MBCP+®, microstructured collagen membrane and total bone marrow grafting: An experimental study in rabbits

The bone tissue engineering models used today are still a long way from any oncologic application as immediate postimplantation irradiation would decrease their osteoinductive potential. The aim of this study was to reconstruct a segmental critical size defect in a weight-bearing bone irradiated after implantation. Six white New Zealand rabbits were immediately implanted with a biomaterial associating resorbable collagen membrane EZ® filled and micro-macroporous biphasic calcium phosphate granules (MBCP+®). After a daily schedule of radiation delivery, and within 4 weeks, a total autologous bone marrow (BM) graft was injected percutaneously into the center of the implant. All the animals were sacrificed at 16 weeks. Successful osseous colonization was found to have bridged the entire length of the defects. Identical distribution of bone ingrowth and residual ceramics at the different levels of the implant suggests that the BM graft plays an osteoinductive role in the center of the defect. Periosteum-like formation was observed at the periphery, with the collagen membrane most likely playing a role. This model succeeded in bridging a large segmental defect in weight-bearing bone with immediate postimplantation fractionated radiation delivery. This has significant implications for the bone tissue engineering approach to patients with cancer-related bone defects

Beta-tricalcium phosphate ceramic triggers fast and robust bone formation by human mesenchymal stem cells

Due to their osteoconductive and inductive properties, a variety of calcium phosphate (CaP) scaffolds are commonly used in orthopaedics as graft material to heal bone defects. In this study, we have used two CaP scaffolds with different hydroxyapatite (HA) and \u3b2-tricalcium phosphate (\u3b2-TCP) ratios (MBCP\uae; 60/40 and MBCP+\uae; 20/80) to investigate their intrinsic capacity to favour human bone marrow stem cells (hBMSCs) osteogenic differentiation capacity. We report that MBCP+\uae showed in in vitro culture model a higher rate of calcium ion release in comparison with MBCP\uae. In two defined coculture systems, the hBMSC seeded onto MBCP+\uae presented an increased amount of VEGF secretion, resulting in an enhanced endothelial cell proliferation and capillary formation compared with hBMSC seeded onto MBCP\uae. When both ceramics combined with hBMSC were implanted in a nude mouse model, we observed a faster osteogenic differentiation and enhancement mature bone deposition sustained by the presence of a vast host vasculature within the MBCP+\uae ceramics. Bone formation was observed in samples highly positive to the activation of calcium sensing receptor protein (CaSr) on the surface of seeded hBMSC that also shown higher BMP-2 protein expression. With these data we provide valuable insights in the possible mechanisms of ossification and angiogenesis by hBMSC that we believe to be primed by calcium ions released from CaP scaffolds. Evidences could lead to an optimization of ceramic scaffolds to prime bone repair

Biofunctionality of MBCP ceramic granules (TricOs™) plus fibrin sealant (Tisseel®) versus MBCP ceramic granules as a filler of large periprosthetic bone defects: an investigative ovine study

We aimed to quantify bone colonization toward an untreated titanium implant with primary stability following filling of the defect with micromacroporous biphasic calcium phosphate (MBCP) granules (TricOs) or MBCP granules mixed with fibrin sealant (Tisseel). Medial arthrotomy was performed on the knees of 20 sheep to create a bone defect (16 mm deep; 10 mm diameter), followed by anchorage of a titanium screw. Defects were filled with TricOs or TricOs-Tisseel granules, a perforated MBCP washer, a titanium washer and titanium screw. Sheep were euthanized at 3, 6, 12 and 26 weeks. From Week 12 onwards, the percentage of bone in contact with the 8 mm anchorage part of the screw increased in both groups, confirming its primary stability. At 26 weeks, whereas bone colonization was similar in both groups, biodegradation of ceramic was more rapid in the TricOs-Tisseel group (P = 0.0422). The centripetal nature of bone colonization was evident. Bone contact with the titanium implant surface was negligible. In conclusion, the use of a model that reproduces a large metaphyseal bone defect around a titanium implant with primary stability, filled with a mixture of either TricOs ceramic granules or TricOs granules mixed with Tisseel fibrin sealant, suggests that the addition of fibrin to TricOs enhances bone filling surgical technology