Physico-Chemical Characteristics and Posterolateral Fusion Performance of Biphasic Calcium Phosphate with Submicron Needle-Shaped Surface Topography Combined with a Novel Polymer Binder.

A biphasic calcium phosphate with submicron needle-shaped surface topography combined with a novel polyethylene glycol/polylactic acid triblock copolymer binder (BCP-EP) was investigated in this study. This study aims to evaluate the composition, degradation mechanism and bioactivity of BCP-EP in vitro, and its in vivo performance as an autograft bone graft (ABG) extender in a rabbit Posterolateral Fusion (PLF) model. The characterization of BCP-EP and its in vitro degradation products showed that the binder hydrolyses rapidly into lactic acid, lactide oligomers and unaltered PEG (polyethylene glycol) without altering the BCP granules and their characteristic submicron needle-shaped surface topography. The bioactivity of BCP-EP after immersion in SBF revealed a progressive surface mineralization. In vivo, BCP-EP was assessed in a rabbit PLF model by radiography, manual palpation, histology and histomorphometry up to 12 weeks post-implantation. Twenty skeletally mature New Zealand (NZ) White Rabbits underwent single-level intertransverse process PLF surgery at L4/5 using (1) autologous bone graft (ABG) alone or (2) by mixing in a 1:1 ratio with BCP-EP (BCP-EP/ABG). After 3 days of implantation, histology showed the BCP granules were in direct contact with tissues and cells. After 12 weeks, material resorption and mature bone formation were observed, which resulted in solid fusion between the two transverse processes, following all assessment methods. BCP-EP/ABG showed comparable fusion rates with ABG at 12 weeks, and no graft migration or adverse reaction were noted at the implantation site nor in distant organs.

Effect on Rheological Properties and 3D Printability of Biphasic Calcium Phosphate Microporous Particles in Hydrocolloid-Based Hydrogels.

The production of patient-specific bone substitutes with an exact fit through 3D printing is emerging as an alternative to autologous bone grafting. To the success of tissue regeneration, the material characteristics such as porosity, stiffness, and surface topography have a strong influence on the cell-material interaction and require significant attention. Printing a soft hydrocolloid-based hydrogel reinforced with irregularly-shaped microporous biphasic calcium phosphate (BCP) particles (150-500 µm) is an alternative strategy for the acquisition of a complex network with good mechanical properties that could fulfill the needs of cell proliferation and regeneration. Three well-known hydrocolloids (sodium alginate, xanthan gum, and gelatin) have been combined with BCP particles to generate stable, homogenous, and printable solid dispersions. Through rheological assessment, it was determined that the crosslinking time, printing process parameters (infill density percentage and infill pattern), as well as BCP particle size and concentration all influence the stiffness of the printed matrices. Additionally, the swelling behavior on fresh and dehydrated 3D-printed structures was investigated, where it was observed that the BCP particle characteristics influenced the constructs' water absorption, particle diffusion out of the matrix and degradability.

MagnetOs, Vitoss, and Novabone in a Multi-endpoint Study of Posterolateral Fusion: A True Fusion or Not?

STUDY DESIGN: This study was a multi-endpoint analysis of bone graft substitutes implanted as a standalone graft in a clinically relevant Ovine model of instrumented posterolateral spinal fusion (PLF). OBJECTIVE: The objective of this study was to obtain high-quality evidence on the efficacy of commercial bone graft substitutes compared with autograft in instrumented PLF using a state-of-the-art model with a complete range of assessment techniques. SUMMARY OF BACKGROUND DATA: Preclinical and clinical data on the quality of spinal fusions obtained with bone graft substitutes are often limited. Calcium phosphates with submicron topography have shown promising results in PLF, as these are able to induce bone formation in tissues distant from the host bone, which facilitates bony union. METHODS: Nine female, skeletally mature sheep (4-5 y) underwent posterior pedicle screw/rods instrumented PLF at L2-L3 and L4-L5 using the following bone graft materials as a standalone graft per spinal segment: (1) biphasic calcium phosphate with submicron topography (BCP<µm), (2) 45S5 Bioglass (BG), and (3) collagen-ß-tricalcium phosphate with a 45S5 Bioglass adjunct (TCP/BG). Autograft bone (AB) was used as a positive control treatment. Twelve weeks after implantation, the spinal segments were evaluated by fusion assessment (manual palpation, x-ray, micro-computed tomography, and histology), fusion mass volume quantification (micro-computed tomography), range of motion (ROM) testing, histologic evaluation, and histomorphometry. RESULTS: Fusion assessment revealed equivalence between AB and BCP<µm by all fusion assessment methods, whereas BG and TCP/BG led to significantly inferior results. Fusion mass volume was highest for BCP<µm, followed by AB, BG, and TCP/BG. ROM testing determined equivalence for spinal levels treated with AB and BCP<µm, while BG and TCP/BG exhibited higher ROM. Histologic evaluation revealed substantial bone formation in the intertransverse regions for AB and BCP<µm, whereas BG and TCP/BG grafts contained fibrous tissue and minimal bone formation. Histologic observations were supported by the histomorphometry data. CONCLUSIONS: This study reveals clear differences in efficacy between commercially available bone graft substitutes, emphasizing the importance of clinically relevant animal models with multiendpoint analyses for the evaluation of bone graft materials. The results corroborate the efficacy of calcium phosphate with submicron topography, as this was the only material that showed equivalent performance to autograft in achieving spinal fusion.

Efficacy of a synthetic calcium phosphate with submicron surface topography as autograft extender in lapine posterolateral spinal fusion.

Posterolateral spinal fusion (PLF) is a common procedure in orthopedic surgery that is performed to fuse adjacent vertebrae to reduce symptoms related to spinal conditions. In the current study, a novel synthetic calcium phosphate with submicron surface topography was evaluated as an autograft extender in a validated rabbit model of PLF. Fifty-nine skeletally mature New Zealand white rabbits were divided into three groups and underwent single-level intertransverse process PLF at L4-5 using (1) autologous bone graft (ABG) alone or in a 1:1 combination with (2) calcium phosphate granules (ABG/BCPgranules ), or (3) granules embedded in a fast-resorbing polymeric carrier (ABG/BCPputty ). After 6, 9, and 12 weeks, animals were sacrificed and spinal fusion was assessed by manual palpation, Radiographs, micro-CT, mechanical testing (12 weeks only), histology, and histomorphometry. Based on all endpoints, all groups showed a gradual progression in bone formation and maturation during time, leading to solid fusion masses between the transverse processes after 12 weeks. Fusion assessments by manual palpation, radiography and histology were consistent and demonstrated equivalent fusion rates between groups, with high bilateral fusion rates after 12 weeks. Mechanical tests after 12 weeks indicated substantially lower range of motion for all groups, compared to non-operated controls. By histology and histomorphometry, the gradual formation and maturation of bone in the fusion mass was confirmed for each graft type. With these results, we describe the equivalent performance between autograft and a novel calcium phosphate material as an autograft extender in a rabbit model of PLF using an extensive range of evaluation techniques. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2080-2090, 2019.

Osteoclast resorption of beta-tricalcium phosphate controlled by surface architecture.

A resorbable bone graft substitute should mimic native bone in its capacity to support bone formation and be remodeled by osteoclasts (OCl) or other multinucleated cells such as foreign body giant cells (FBGC). We hypothesize that by changing the scale of surface architecture of beta-tricalcium phosphate (TCP), cellular resorption can be influenced. CD14(+) monocyte precursors were isolated from human peripheral blood (n = 4 independent donors) and differentiated into OCl or FBGC on the surface of TCP discs comprising either submicron- or micron-scale surface topographical features (TCPs and TCPb, respectively). On submicrostructured TCPs, OCl survived, fused, differentiated, and extensively resorbed the substrate; however, on microstructured TCPb, OCl survival, TRAP activation, and fusion were attenuated. Importantly, no resorption was observed on microstructured TCPb. By confocal microscopy, OCl formed on TCPs contained numerous actin rings allowing for resorption, but not on TCPb. In comparison, FBGC could not resorb either TCP material, suggesting that osteoclast-specific machinery is necessary to resorb TCP. By tuning surface architecture, it appears possible to control osteoclast resorption of calcium phosphate. This approach presents a useful strategy in the design of resorbable bone graft substitutes.

Liposomal clodronate inhibition of osteoclastogenesis and osteoinduction by submicrostructured beta-tricalcium phosphate.

Bone graft substitutes such as calcium phosphates are subject to the innate inflammatory reaction, which may bear important consequences for bone regeneration. We speculate that the surface architecture of osteoinductive ß-tricalcium phosphate (TCP) stimulates the differentiation of invading monocyte/macrophages into osteoclasts, and that these cells may be essential to ectopic bone formation. To test this, porous TCP cubes with either submicron-scale surface architecture known to induce ectopic bone formation (TCPs, positive control) or micron-scale, non-osteoinductive surface architecture (TCPb, negative control) were subcutaneously implanted on the backs of FVB strain mice for 12 weeks. Additional TCPs samples received local, weekly injections of liposome-encapsulated clodronate (TCPs + LipClod) to deplete invading monocyte/macrophages. TCPs induced osteoclast formation, evident by positive tartrate resistant acid phosphatase (TRAP) cytochemical staining and negative macrophage membrane marker F4/80 immunostaining. No TRAP positive cells were found in TCPb or TCPs + LipClod, only F4/80 positive macrophages and foreign body giant cells. TCPs stimulated subcutaneous bone formation in all implants, while no bone could be found in TCPb or TCPs + LipClod. In agreement, expression of bone and osteoclast gene markers was upregulated in TCPs versus both TCPb and TCPs + LipClod, which were equivalent. In summary, submicron-scale surface structure of TCP induced osteoclastogenesis and ectopic bone formation in a process that is blocked by monocyte/macrophage depletion.

Molecular mechanisms of biomaterial-driven osteogenic differentiation in human mesenchymal stromal cells.

Calcium phosphate (CaP) based ceramics are used as bone graft substitutes in the treatment of bone defects. The physico-chemical properties of these materials determine their bioactivity, meaning that molecular and cellular responses in the body will be tuned accordingly. In a previous study, we compared two porous CaP ceramics, hydroxyapatite (HA) and ß-tricalcium phosphate (TCP), which, among other properties, differ in their degradation behaviour in vitro and in vivo, and we demonstrated that the more degradable ß-TCP induced more bone formation in a heterotopic model in sheep. This is correlated to in vitro data, where human bone marrow derived mesenchymal stromal cells (MSC) exhibited higher expression of osteogenic differentiation markers, such as osteopontin, osteocalcin and bone sialoprotein, when cultured in ß-TCP than in HA. More recently, we also showed that this effect could be mimicked in vitro by exposure of MSC to high concentrations of calcium ions (Ca(2+)). To further correlate surface physico-chemical dynamics of HA and ß-TCP ceramics with the molecular response of MSC, we followed Ca(2+) release and surface changes in time as well as cell attachment and osteogenic differentiation of MSC on these ceramics. Within 24 hours, we observed differences in cell morphology, with MSC cultured in ß-TCP displaying more pronounced attachment and spreading than cells cultured in HA. In the same time frame, ß-TCP induced expression of G-protein coupled receptor (GPCR) 5A and regulator of G-protein signaling 2, revealed by DNA microarray analysis. These genes, associated with the protein kinase A and GPCR signaling pathways, may herald the earliest response of MSC to bone-inducing ceramics.

In vivo performance of microstructured calcium phosphate formulated in novel water-free carriers.

Osteoinductive calcium phosphate (CaP) ceramics can be combined with polymeric carriers to make shapeable bone substitutes as an alternative to autologous bone; however, carriers containing water may degrade the ceramic surface microstructure, which is crucial to bone formation. In this study five novel tricalcium phosphate (TCP) formulations were designed from water-free polymeric binders and osteoinductive TCP granules of different particle sizes (500-1000 µm for moldable putty forms, and 150-500 µm for flowable paste forms). The performance of these novel TCP formulations was studied and compared with control TCP granules alone (both 150-500 and 500-1000 µm). In vitro the five TCP formulations were characterized by their carrier dissolution times and TCP mineralization kinetic profiles in simulated body fluid. In vivo the formulations were implanted in the dorsal muscle and a unicortical femoral defect (Ø=5 mm) of dogs for 12 weeks. The TCP formulation based on a xanthan gum-glycerol carrier exhibited fast carrier dissolution (1 h) and TCP mineralization (7 days) in vitro, but induced inflammation and showed little ectopic bone formation. This carrier chemistry was thus found to disrupt the early cellular response related to osteoinduction by microstructured TCP. TCP formulations based on carboxymethyl cellulose-glycerol and Polyoxyl 15-hydroxystearate-Pluronic(®) F127 allowed the in vitro surface mineralization of TCP by day 7 and produced the highest level of orthotopic bone bridging and ectopic bone formation, which was equivalent to the control. These results demonstrate that water-free carriers can preserve the chemistry, microstructure, and performance of osteoinductive CaP ceramics.

The effects of inorganic additives to calcium phosphate on in vitro behavior of osteoblasts and osteoclasts.

This study describes a medium-throughput system based on deposition of calcium phosphate films in multi-well tissue culture plates that can be used to study the effect of inorganic additives on the behavior of osteoblasts and osteoclasts in a standardized manner. All tested elements, copper, zinc, strontium, fluoride and carbonate were homogenously deposited into calcium phosphate films in varying concentrations by using a biomimetic approach. The additives affected morphology and composition of calcium phosphate films to different extent, depending on the concentration used. The effect on proliferation and differentiation of MC3T3-E1 osteoblasts depended on the compound and concentration tested. In general, copper and zinc ions showed an inhibitory effect on osteoblast proliferation, the effect of strontium was concentration dependent, whereas films containing fluoride and carbonate, respectively, augmented osteoblast proliferation. Copper and zinc had no effect or were mild inhibitory on osteoblast differentiation, while strontium, fluoride and carbonate ions demonstrated a clear decrease in differentiation in comparison to the control films without additives. Primary osteoclasts cultured on calcium phosphate films containing additives showed a significantly decreased resorptive activity as compared to the control, independent on the element incorporated. No cytotoxic effect of the elements in the concentrations tested was observed. The system presented in this study mimics bone mineral containing trace elements, making it useful for studying fundamental processes of bone formation and turnover. The present results can be used for modifying bone graft substitutes by addition of inorganic additives in order to affect their performance in bone repair and regeneration.