Osteogenic Effect of a Bioactive Calcium Alkali Phosphate Bone Substitute in Humans.

(1) Background: The desire to avoid autograft harvesting in implant dentistry has prompted an ever-increasing quest for bioceramic bone substitutes, which stimulate osteogenesis while resorbing in a timely fashion. Consequently, a highly bioactive silicon containing calcium alkali orthophosphate (Si-CAP) material was created, which previously was shown to induce greater bone cell maturation and bone neo-formation than ß-tricalcium phosphate (ß-TCP) in vivo as well as in vitro. Our study tested the hypothesis that the enhanced effect on bone cell function in vitro and in sheep in vivo would lead to more copious bone neoformation in patients following sinus floor augmentation (SFA) employing Si-CAP when compared to ß-TCP. (2) Methods: The effects of Si-CAP on osteogenesis and Si-CAP resorbability were evaluated in biopsies harvested from 38 patients six months after SFA in comparison to ß-TCP employing undecalcified histology, histomorphometry, and immunohistochemical analysis of osteogenic marker expression. (3) Results: Si-CAP as well as ß-TCP supported matrix mineralization and bone formation. Apically furthest away from the original bone tissue, Si-CAP induced significantly higher bone formation, bone-bonding (bone-bioceramic contact), and granule resorption than ß-TCP. This was in conjunction with a higher expression of osteogenic markers. (4) Conclusions: Si-CAP induced higher and more advanced bone formation and resorbability than ß-TCP, while ß-TCP's remarkable osteoconductivity has been widely demonstrated. Hence, Si-CAP constitutes a well-suited bioactive graft choice for SFA in the clinical arena.

A tissue engineered 3D printed calcium alkali phosphate bioceramic bone graft enables vascularization and regeneration of critical-size discontinuity bony defects in vivo.

Introduction: Recently, efforts towards the development of patient-specific 3D printed scaffolds for bone tissue engineering from bioactive ceramics have continuously intensified. For reconstruction of segmental defects after subtotal mandibulectomy a suitable tissue engineered bioceramic bone graft needs to be endowed with homogenously distributed osteoblasts in order to mimic the advantageous features of vascularized autologous fibula grafts, which represent the standard of care, contain osteogenic cells and are transplanted with the respective blood vessel. Consequently, inducing vascularization early on is pivotal for bone tissue engineering. The current study explored an advanced bone tissue engineering approach combining an advanced 3D printing technique for bioactive resorbable ceramic scaffolds with a perfusion cell culture technique for pre-colonization with mesenchymal stem cells, and with an intrinsic angiogenesis technique for regenerating critical size, segmental discontinuity defects in vivo applying a rat model. To this end, the effect of differing Si-CAOP (silica containing calcium alkali orthophosphate) scaffold microarchitecture arising from 3D powder bed printing (RP) or the Schwarzwalder Somers (SSM) replica fabrication technique on vascularization and bone regeneration was analyzed in vivo. In 80 rats 6-mm segmental discontinuity defects were created in the left femur. Methods: Embryonic mesenchymal stem cells were cultured on RP and SSM scaffolds for 7d under perfusion to create Si-CAOP grafts with terminally differentiated osteoblasts and mineralizing bone matrix. These scaffolds were implanted into the segmental defects in combination with an arteriovenous bundle (AVB). Native scaffolds without cells or AVB served as controls. After 3 and 6 months, femurs were processed for angio-µCT or hard tissue histology, histomorphometric and immunohistochemical analysis of angiogenic and osteogenic marker expression. Results: At 3 and 6 months, defects reconstructed with RP scaffolds, cells and AVB displayed a statistically significant higher bone area fraction, blood vessel volume%, blood vessel surface/volume, blood vessel thickness, density and linear density than defects treated with the other scaffold configurations. Discussion: Taken together, this study demonstrated that the AVB technique is well suited for inducing adequate vascularization of the tissue engineered scaffold graft in segmental defects after 3 and 6 months, and that our tissue engineering approach employing 3D powder bed printed scaffolds facilitated segmental defect repair.

Development of a synthetic tissue engineered three-dimensional printed bioceramic-based bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro.

Over the last decade there have been increasing efforts to develop three-dimensional (3D) scaffolds for bone tissue engineering from bioactive ceramics with 3D printing emerging as a promising technology. The overall objective of the present study was to generate a tissue engineered synthetic bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro, thereby mimicking the advantageous properties of autogenous bone grafts and facilitating usage for reconstructing segmental discontinuity defects in vivo. To this end, 3D scaffolds were developed from a silica-containing calcium alkali orthophosphate, using, first, a replica technique - the Schwartzwalder-Somers method - and, second, 3D printing, (i.e. rapid prototyping). The mechanical and physical scaffold properties and their potential to facilitate homogenous colonization by osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture were examined. Osteoblastic cells were dynamically cultured for 7 days on both scaffold types with two different concentrations of 1.5 and 3 × 109 cells/l. The amount of cells and bone matrix formed and osteogenic marker expression were evaluated using hard tissue histology, immunohistochemical and histomorphometric analysis. 3D-printed scaffolds (RPS) exhibited more micropores, greater compressive strength and silica release. RPS seeded with 3 × 109 cells/l displayed greatest cell and extracellular matrix formation, mineralization and osteocalcin expression. In conclusion, RPS displayed superior mechanical and biological properties and facilitated generating a tissue engineered synthetic bone graft in vitro, which mimics the advantageous properties of autogenous bone grafts, by containing homogenously distributed terminally differentiated osteoblasts and mineralizing bone matrix and therefore is suitable for subsequent in vivo implantation for regenerating segmental discontinuity bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

Osteoclastic bioresorption of biomaterials: two- and three-dimensional imaging and quantification.

PURPOSE: Bioresorbable materials have been developed in the hope that the body will replace them with newly formed tissue. The first step of this remodeling process in bone is the bioresorption of the material by osteoclasts. The aim of this study was to analyze osteoclastic resorption of biomaterials in vitro using the commonly used two-dimensional methods of light-microscopy (LM) and scanning electron microscopy (SEM) in comparison with infinite focus microscopy (IFM), a recently developed imaging method allowing for three-dimensional surface analysis. METHODS: Human hematopoietic stem cells were cultivated in the presence of the cytokines M-CSF and RANK-L for 4 weeks directly on dentin and a calcium phosphate cement. Osteoclast development was surveyed with standard techniques. After removal of the cells, resorption was characterized and quantified by LM, SEM and IFM. RESULTS: Osteoclast cultures on the biomaterials presented the typical osteoclast-specific markers. On dentin samples LM, SEM as well as IFM allowed for discrimination of resorption. Quantification of the resorbed area showed a linear correlation between the results (LM vs. SEM: r=0.996, p=0.004; SEM vs. IFM: r=0.989, p=0.011; IFM vs. LM: r=0.995). It was not possible to demarcate resorption pits on GB14 using LM or SEM. With IFM, resorption on GB14 could be visualized and quantified two- and three-dimensionally. CONCLUSIONS: In this paper we introduce IFM as a technology for three-dimensional visualization and quantification of resorption of biomaterials. Better understanding of the bioresorption of biomaterials may help in the design of better materials and might therefore constitute an important step on the avenue to the development of artificial bone.

Femtosecond laser induced fixation of calcium alkali phosphate ceramics on titanium alloy bone implant material.

Femtosecond lasers provide a novel method of attaching bioceramic material to a titanium alloy, thereby improving the quality of bone implants. The ultrashort 30 fs laser pulses (790 nm wavelength) penetrate a thin dip-coated layer of fine ceramic powder, while simultaneously melting a surface layer of the underlying metal. The specific adjustment of the laser parameters (pulse energy and number of pulses per spot) avoids unnecessary melting of the bioactive calcium phosphate, and permits a defined thin surface melting of the metal, which in turn is not heated throughout, and therefore maintains its mechanical stability. It is essential to choose laser energy densities that correspond to the interval between the ablation fluences of both materials involved: about 0.1-0.4 Jcm(-2). In this work, we present the first results of this unusual technique, including laser ablation studies, scanning electron microscopy and optical microscope images, combined with EDX data.

Inhibition of mineralization by a calcium zirconium phosphate coating.

Bioactive ceramics used as coating materials combine the conductive properties of a bioceramic with the mechanical stability of the metal implant. We studied a calcium zirconium phosphate-containing coating material, FA-CZP [Ca(5)(PO(4))(3)F, CaZr(4)(PO(4))(6)], that is relatively insoluble in the biological milieu. The reaction of bone to this material was investigated histologically and histomorphometrically in an animal trial. Cylindrical Ti6Al4V specimens that had been coated with FA-CZP by plasma spraying were implanted in the femoral condyles of rabbits. The implants were left in place for 2, 4, 6, 12, and 14 weeks. FA-CZP led to impaired mineralization of the newly formed bone at the interface. Noncalcified osteoid was found throughout the whole study period. The layer seemed to become thicker with time. The mineralization disorder is evidently caused by zirconium ions. The presence of zirconium in the osteoid in contact with the implant was demonstrated by means of two different staining methods.

Histological and histomorphometric investigations on bone integration of rapidly resorbable calcium phosphate ceramics.

Resorbable ceramics can promote the bony integration of implants. Their rate of degradation should ideally be synchronized with bone regeneration. We report here the results of a histological study of implants with two resorbable calcium phosphate ceramic coatings: Ca(2)KNa(PO(4))(2)-(GB14) and Ca(10)[K/Na](PO(4))(7)-(602020). The results attained with these ceramic-coated implants show the benefits of these materials with regard to bioactive bone-healing stimulation, compared with uncoated implants. The GB14 ceramic coating exhibited greater bone regeneration and differentiation on its surface than the conventional hydroxyapatite coating and helped bone tissue achieve more extensive contact free of connective tissue. Not until the coating disintegrated did the histological features of GB14- and 602020-coated implants converge-both implant types were integrated into bone. Rapid disintegration of the coating material, as with 602020, supports osteoblast proliferation but has negative effects on bone mineralization. Both resorbable ceramics tested, GB14 and 602020, demonstrated bioactivity; even metal surfaces coated with these materials were populated by mature bone tissue without connective tissue after disintegration of their ceramic coating. The less rapidly degrading material, GB14, achieved better results. Degradable calcium phosphate coatings have the potential to stimulate bone regeneration. From the histological viewpoint, the resorbable ceramics examined here can be recommended as coating materials for clinical use.

The effect of bioactive glass ceramics on the expression of bone-related genes and proteins in vitro.

Using biodegradable bone substitutes in alveolar ridge augmentation avoids second-site surgery for autograft harvesting. Considerable efforts have been undertaken to develop rapidly resorbable bone substitute materials with a higher degree of biodegradability than tricalcium phosphate (TCP). This study examines the effect of novel biodegradable glass ceramics on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior with that of TCP. Test materials used were alpha-TCP, a surface-treated glass ceramic GB9N with crystalline phase Ca(2)KNa(PO(4))(2) and a small amount of amorphous silica phosphate; AP40 - a glass ceramic based on crystalline phases of apatite and wollastonite; and a glass ceramic Mg5 composed of 20.6% CaO, 58.5% P(2)O(5), 14.4% Na(2)O, 4.1% MgO and 2.4% CaF(2) (wt%). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various bone-related mRNAs and proteins (type I collagen (Col I), osteocalcin (OC), osteopontin (OP), osteonectin (ON), alkaline phosphatase (ALP) and bone sialoprotein (BSP)). The substrata supported continuous cellular growth for 21 days. By day 21, GB9N had the highest number of HBDC. GB9N induced significantly enhanced expression of Col I, ALP, OP, OC and ON mRNA at 3 days; of OP, OC and ON mRNA and protein at 7 and 14 days; and of ALP, OP and OC mRNA and Col I, ALP, BSP, ON and OP protein at 21 days. Since all novel glass ceramics supported cellular proliferation together with expression of bone-related genes and proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. GB9N had the most effect on osteoblastic differentiation, thus suggesting that this material may possess a higher potency to enhance osteogenesis than TCP.