Calcium Phosphate Granules Research Articles

Endochondral ossification: insights into the cartilage mineralization processes achieved by an anhydrous freeze substitution protocol

Growth plate cartilage (GP) serves as a dynamic site of active mineralization and offers a unique opportunity to investigate the cell-regulated matrix mineralization process. Transmission electron microscopy (TEM) provides a means for the direct observation of these mechanisms, offering the necessary resolution and chemical analysis capabilities. However, as mineral crystallinity is prone to artifacts using aqueous fixation protocols, sample preparation techniques are critical to preserve the mineralized tissue in its native form. We optimized cryofixation by high-pressure freezing followed by freeze substitution in anhydrous acetone containing 0.5% uranyl acetate to prepare murine GP for TEM analysis. This sample preparation workflow maintains cellular and extracellular protein structural integrity with sufficient contrast for observation and without compromising mineral crystallinity. By employing appropriate sample preparation techniques, we were able to observe two parallel mineralization processes driven by chondrocytes: 1) intracellular- and 2) extracellular-originating mineralized vesicles. Both mechanisms are based on sequestering calcium phosphate (CaP) within a membrane-limited structure, albeit originating from different compartments of the chondrocytes. In the intracellular originating pathway, CaP accumulates within mitochondria as globular CaP granules, which are incorporated into intracellular vesicles (500-1000 nm) and transported as granules to the extracellular matrix (ECM). In contrast, membrane budding vesicles with a size of approximately 100-200nm, filled with needle-shaped minerals were observed only in the ECM. Both processes transport CaP to the collagenous matrix via vesicles, they can be differentiated based on the vesicle size and mineral morphologies. Their individual importance to the cartilage mineralization process is yet to be determined. Statement of SignificanceWe do not fully understand the process by which epiphyseal cartilage mineralizes - a vital step in endochondral bone formation. Previous work has proposed that mitochondria and intracellular vesicles are storage sites for the delivery of mineral to collagen fibrils. However, these concepts are founded on results from in vitro models of mineralization; no prior work has observed mineral-containing intracellular vesicles or mitochondria in developing epiphyseal cartilage. Here we developed a new cryofixation preparation route for transmission electron microscopy (TEM) imaging that has disclosed a cell-regulated process of mineralization in epiphyseal cartilage. High resolution TEM images revealed an involvement of mitochondria and intracellular and extracellular vesicles in delivering transient mineral phases to the collagen fibrils to promote cartilage mineralization.

Fabrication of magnesium-doped biphasic calcium phosphate granules with sea urchin spine-derived porous structure

In bone regeneration, synthetic materials are required to replace autogenous bone grafts. The shape, composition, surface, and porous structure of the material are key factors in bone formation and material resorption. In this study, we describe the fabrication of Mg-doped biphasic calcium phosphate (Mg-BCP) granules comprising hydroxyapatite (HAp) and beta-tricalcium phosphate (β-TCP) by exploiting the structural and chemical characteristics of sea urchin spines. The Mg-BCP granules were cylinders with a 1.2–1.8 mm diameter and height of 1.4–1.7 mm. The β-TCP/HAp ratio and Mg content in the Mg-BCP granules were controlled within the ranges of 0.36–0.45 and 2.40–6.21 mol%, respectively. Controlling the β-TCP/HAp ratio and Mg content may contribute to controlling the material resorption rate and promoting osteogenesis and angiogenesis. The Mg-BCP granules maintained the structural characteristics of the sea urchin spines. The granules comprised radial wedges termed septa, and a meshwork termed stereom. The interseptal gaps, or micropores, decreased from the granular exterior (24–29 μm) to the interior (14–28 μm). The septa in the outermost layer provide concavities on the sides of the cylindrical granules. The stereom meshworks in the core and outer layers involved micropores in the ranges of 3–15 and 3–10 μm, respectively. These multiscale pores and surface undulations may improve cell penetration and attachment, bodily fluid permeability, and protein adsorption. The compressive strengths of the Mg-BCP granules were in the range of 45.2–46.1 MPa, which are useful as bone grafts. Due to their rounded shapes, the Mg-BCP granules filled a mold more densely than the clinically used and irregularly shaped granules. These findings demonstrated that Mg-BCP granules are promising materials for bone regeneration.

Incorporating strontium enriched amorphous calcium phosphate granules in collagen/collagen-magnesium-hydroxyapatite osteochondral scaffolds improves subchondral bone repair

Osteochondral defect repair with a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold has demonstrated good clinical results. However, subchondral bone repair remained suboptimal, potentially leading to damage to the regenerated overlying neocartilage. This study aimed to improve the bone repair potential of this scaffold by incorporating newly developed strontium (Sr) ion enriched amorphous calcium phosphate (Sr-ACP) granules (100–150 μm). Sr concentration of Sr-ACP was determined with ICP-MS at 2.49 ± 0.04 wt%. Then 30 wt% ACP or Sr-ACP granules were integrated into the scaffold prototypes. The ACP or Sr-ACP granules were well embedded and distributed in the collagen matrix demonstrated by micro-CT and scanning electron microscopy/energy dispersive x-ray spectrometry. Good cytocompatibility of ACP/Sr-ACP granules and ACP/Sr-ACP enriched scaffolds was confirmed with in vitro cytotoxicity assays. An overall promising early tissue response and good biocompatibility of ACP and Sr-ACP enriched scaffolds were demonstrated in a subcutaneous mouse model. In a goat osteochondral defect model, significantly more bone was observed at 6 months with the treatment of Sr-ACP enriched scaffolds compared to scaffold-only, in particular in the weight-bearing femoral condyle subchondral bone defect. Overall, the incorporation of osteogenic Sr-ACP granules in Col/Col-Mg-HAp scaffolds showed to be a feasible and promising strategy to improve subchondral bone repair.

Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H2O2 to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.

Bone formation beyond the skeletal envelope using calcium phosphate granules packed into a collagen pouch—a pilot study

In this proof-of-concept, bone neoformation beyond the skeletal envelope is explored by using a collagen pouch (n = 6) packed with calcium phosphate (CaP) granules placed over the frontal bone in sheep (n = 3). At 13 weeks, macroscopic examination showed specimens covered by an adherent fibrinous envelope with slight vascularization. Histology revealed colonization of the implant by newly formed woven bone and fibrous connective tissue. Surface osteoblasts as well as material-filled macrophages, lymphocytes, polymorphonuclear cells and giant cells were also found in large quantities surrounding the newly formed bone tissue inside the collagen pouch. On the side facing the recipient bone, the collagen membrane had to a large extent been resorbed and bridging bone formation was clearly visible between the test article and recipient bone. On the other side facing soft tissue, the collagen pouch remained intact with a visible fibrous capsule. This study demonstrated that the use of a collagen sleeve as a container for CaP granules allows for good neoformation beyond the skeletal envelope with bridging bone formation clearly visible between the test article and recipient bone. Additionally, in this model, macrophages rather than osteoclasts appear to modulate CaP granule resorption and remodeling into new bone. This construct opens new perspectives for treatment methods that could be used for bone augmentation and restoration of cranio-maxillofacial defects and malformations.

Enabling efficient phosphorus recovery from cow manure: Liberation of phosphorus through acidification and recovery of phosphorus as calcium phosphate granules

Cow manure (CM) is one of the largest secondary sources of phosphorus, while mined phosphorus rock is becoming scarce and diminishing in quality. Phosphorus recovery from CM is essential for increasing the phosphorus use efficiency and, therefore, securing future food production. Calcium phosphate (CaP) granulation can enable efficient phosphorus recovery during anaerobic digestion. However, in CM, the absence of phosphorus in the ionic form and the high presence of inorganic carbon and magnesium inhibits CaP granulation. In this study, CaP granulation was achieved for the first time during CM treatment, using an up-flow anaerobic sludge blanket (UASB) reactor preceded by an acidifying continuously stirred tank reactor (CSTR). The UASB reactor and CSTR ran for 190 days at organic loading rates of 1 and 22.5 gCOD Lreactor-1 d-1, respectively. The acidification of CM at pH 5 dissolved struvite and calcium carbonate, causing > 94% of phosphorus, calcium and magnesium to be present as ions. In the UASB reactor fed with the CSTR effluent, 83 ± 3% of phosphorus was removed and mainly present as CaP granules > 2.5 mm in diameter. The CaP granules had a phosphorus content of 8.4 ± 1.3%P w/w with amorphous and crystalline calcium phosphate being the dominant phases. The formation of large and concentrated CaP granules causes accumulation at the bottom of the reactor and allows for simple size separation leading to efficient phosphorus recovery The recovery of CaP granules improves CM valorization and can reduce the transport of manure by up to 87%. The acidification prior to digestion thus promotes a more efficient use of phosphorus from CM in agriculture and can replace phosphorus from primary phosphorus sources.

Injectable Hydrogel Membrane for Guided Bone Regeneration.

In recent years, multicomponent hydrogels such as interpenetrating polymer networks (IPNs) have emerged as innovative biomaterials due to the synergistic combination of the properties of each network. We hypothesized that an innovative non-animal IPN hydrogel combining self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) with photochemically cross-linkable dextran methacrylate (DexMA) could be a valid alternative to porcine collagen membranes in guided bone regeneration. Calvaria critical-size defects in rabbits were filled with synthetic biphasic calcium phosphate granules in conjunction with Si-HPMC; DexMA; or Si-HPMC/DexMA experimental membranes; and in a control group with a porcine collagen membrane. The synergistic effect obtained by interpenetration of the two polymer networks improved the physicochemical properties, and the gel point under visible light was reached instantaneously. Neutral red staining of murine L929 fibroblasts confirmed the cytocompatibility of the IPN. At 8 weeks, the photo-crosslinked membranes induced a similar degree of mineral deposition in the calvaria defects compared to the positive control, with 30.5 ± 5.2% for the IPN and 34.3 ± 8.2% for the collagen membrane. The barrier effect appeared to be similar in the IPN test group compared with the collagen membrane. In conclusion, this novel, easy-to-handle and apply, photochemically cross-linkable IPN hydrogel is an excellent non-animal alternative to porcine collagen membrane in guided bone regeneration procedures.

Preparation and evaluation of osteoinductive porous biphasic calcium phosphate granules obtained from eggshell for bone tissue engineering

The synthetic bone graft substitutes currently used clinically are osteoconductive but not osteoinductive; their low success rate is thus their biggest disadvantage. The use of biomass raw materials for synthesizing calcium phosphate has gradually attracted increased research attention. In this study, hydroxyapatite powder was prepared through liquid-phase precipitation from eggshell, and porous biphasic calcium phosphate granules (EBGs) were then obtained using a pore former and sintering procedure. The EBGs were discovered to have high biocompatibility and no cytotoxicity. The results of animal experiments showed that the area of new bone growth was high, numerous Haversiancanals could be observed, and almost all EBGs were surrounded by new bone tissue, which proved that the EBGs had excellent osteoinductivity. By contrast, numerous fat cells were found in the femoral defect area when a commercial bone graft was employed. Various biological inorganic ions (Mg, Sr, Na, and Fe) originally in the eggshell raw materials were incorporated into the EBGs, and the EBGs exhibited excellent osteogenic abilities. The developed approach provides an economical and feasible solution for the treatment of bone defects.

Effects of cooling conditions and chitosan coating on the properties of porous calcium phosphate granules produced from hard clam shells

• Clam shell was used to synthesize CaP granules by gas foaming technique. • Samples under furnace cooling condition were biphasic CaP granules (HA/β-TCP). • Samples under air cooling condition were triphasic CaP granules (HA/β-TCP/α-TCP). • Samples air-cooled and chitosan-coated had maximum compressive strength. The main inorganic components of clam shells are calcium carbonate and trace elements of magnesium, strontium, and zinc. Clam shells can be used as a calcium source to synthesize calcium phosphates, and these trace elements promote the growth of bone tissue and improve the performance of bioceramics. In this study, hydroxyapatite (HA) powders were synthesized from clam shells, and porous calcium phosphate granules were prepared through the gas foaming technique. In addition, the effects of a chitosan coating and cooling conditions on the strength of the porous granules were investigated. The results indicated that the samples produced under the furnace cooling condition were biphasic hydroxyapatite/β-tricalcium phosphate granules (HA/β-TCP), whereas the samples produced under the air-cooling condition were triphasic hydroxyapatite/β-tricalcium phosphate/α-tricalcium phosphate granules (HA/β-TCP/α-TCP). The compressive strength of the porous granules prepared through air cooling was 79% higher than that of the granules produced through furnace cooling. The compressive strength of the air-cooled sample after the subsequent application of the chitosan coating further increased by 21%. A degradability test revealed that the weight loss rate of the air-cooled samples was greater than that of the furnace-cooled samples, which was due to the presence of high-solubility α-TCP in the air-cooled samples.

Calcium phosphate cements comprising spherical porous calcium phosphate granules: synthesis, structure, and properties

ABSTRACT Calcium phosphate cements (CPCs) are used as artificial bone materials. The bone regeneration ability of CPCs can be improved by controlling their composition, porosity, and pore size. This study aims to design novel CPCs with high bone regeneration ability by controlling their microstructure. CPCs with macropores and micropores were prepared by incorporating spherical porous calcium phosphate granules composed of rod-shaped, calcium-deficient hydroxyapatite (CDHA) or plate-shaped octacalcium phosphate (OCP) particles. The granules were mixed with a binder (cement powder) composed primarily of α-tricalcium phosphate. The structure, morphology, compressive strength, porosity, specific surface area, pore size distribution, dissolution characteristics, and effects on cell viabilities were studied for the synthesized samples. The CPCs composed of porous granules had high porosity (~80%) and both macropores and micropores, which are expected to contribute to bone regeneration. The CPCs composed of porous granules showed a smaller specific surface area but a larger dissolution rate than the granule-free samples. The CPC composed of OCP granules showed a higher dissolution rate than the CPCs containing CDHA granules. In the cell culture experiments, the preosteoblasts proliferated on the CPCs, indicating that these CPCs could function as scaffolds for bone regeneration.

Synthesis of cross-linked chitosan by calcium phosphate as long-term drug delivery coating with cytocompatibility

Antimicrobial therapy was highly efficient at peri-implant infection; however, the in vitro study indicated that high doses of drug had adverse effect on cells. In this study, cefazolin-containing coatings on titanium with cytocompatibility and sustainable release properties using physically cross-linked chitosan as the drug carrier were synthesized. Two drug carrier coatings, cefazolin/chitosan (P-Z@C) and cefazolin/chitosan cross-linked by calcium phosphate (P-Z@C/CP), were electrophoretically deposited on titanium using pulsed direct current (DC) power. The microstructures, drug loadings, drug release rates, antibacterial abilities, and cytotoxicities of the coatings were investigated. Transmission electron microscopy images showed that the calcium phosphate granules were distributed in a chitosan matrix in the P-Z@C/CP coating, and the calcium phosphate granules had a lower drug concentration than that of the surrounding chitosan. The drug content of the P-Z@C coating (259.6 μg/cm2) was approximately thrice that of the P-Z@C/CP coating (95.4 μg/cm2). However, the drug release rate of the P-Z@C coating (75.0 %) was slower than that of the P-Z@C/CP coating (85.9 %) after 30 days. The high sustainable drug-release ability of the P-Z@C coating is attributed to its low swelling ratio. Furthermore, there was no significant difference in the cell numbers between the P-Z@C/CP coating and titanium, but the P-Z@C coating had decreased cell numbers comparing with titanium. After 24 h, all the drug-containing coatings exhibited good antibacterial activity against Staphylococcus aureus (S. aureus).

Structure Detection in Three-Dimensional Cellular Cryoelectron Tomograms by Reconstructing Two-Dimensional Annotated Tilt Series.

The revolutionary technique cryoelectron tomography (cryo-ET) enables imaging of cellular structure and organization in a near-native environment at submolecular resolution, which is vital to subsequent data analysis and modeling. The conventional structure detection process first reconstructs the three-dimensional (3D) tomogram from a series of two-dimensional (2D) projections and then directly detects subcellular components found within the tomogram. However, this process is challenging due to potential structural information loss during the tomographic reconstruction and the limited scope of existing methods since most major state-of-the-art object detection methods are designed for 2D rather than 3D images. Therefore, in this article, as an alternative approach to complement the conventional process, we propose a novel 2D-to-3D framework that detects structures within 2D projection images before reconstructing the results back to 3D. We implemented the proposed framework as three specific algorithms for three individual tasks: semantic segmentation, edge detection, and object localization. As experimental validation of the 2D-to-3D framework for cryo-ET data, we applied the algorithms to the segmentation of mitochondrial calcium phosphate granules, detection of spherical edges, and localization of mitochondria. Quantitative and qualitative results show better performance for prediction tasks of segmentation on the 2D projections and promising performance on object localization and edge detection, paving the way for future studies in the exploration of cryo-ET for in situ structural biology.

Bone morphogenetic protein-2 incorporated calcium phosphate graft promotes peri-implant bone defect healing in dogs: A pilot study.

ObjectivesThe evaluation of three different drug delivery modes of bone morphogenetic protein‐2 (BMP‐2) in healing peri‐implant bone defects in beagle dogs. BMP‐2 was incorporated in or onto calcium phosphate (CaP) granules in various ways: (i) directly on the outer layer of granules CaP: as an adsorbed depot; (ii) during the entire precipitation process of CaP: an internally incorporated depot; or (iii) during the biomimetic coating precipitation of BMP‐2 on the surface of CaP granules: as a coating incorporated depot.Material and MethodsAfter extraction of the lower molars and wound healing in 6 male beagle dogs, 36 implants were placed (n = 6 animal per group). Peri‐implant bone defects were induced. The following treatment groups were evaluated: no treatment; air abrasive surface cleaning (SC) using hydroxyapatite; SC and the subsequent filling of the defect with CaP without BMP‐2; SC plus the subsequent filling of the defect with CaP adsorbed BMP‐2; SC plus the subsequent filling of the defect with CaP internally incorporated BMP‐2; SC plus the subsequent filling of the defect with CaP coating incorporated BMP‐2. Histological and histomorphometric analyses were carried out to quantify and compare the changes in bone tissue surrounding the treated implants.ResultsIn Group 1 with no treatment, four implants were lost. Group 5 with the SC and the subsequent filling of the defect with internally incorporated BMP‐2 biomimetically prepared CaP (BioCaP), whereby the BMP‐2 is incorporated in the entire volume of all BioCaP particles, showed overall the best results to regenerate bone around the implants.ConclusionThis study concluded that the group treated with SC plus the subsequent filling of the defect with CaP BMP‐2 internally incorporated BMP‐2, whereby BMP‐2 has been incorporated in the entire volume of all CaP particles, showed overall the best results when aiming to regenerate bone around the implants.

Treatment of long bone infection by a biodegradable bone cement releasing antibiotics in human.

Repair of methicillin-resistant Staphylococcal (MRSA) chronic osteomyelitis and resulting bone defect is one of the major challenges in orthopaedics. Previous study has shown the effectiveness of antibiotic loaded biodegradable composite bone cement with in vitro tests and in the treatment of experimental osteomyelitis. The cement is composed of poly(lactide-co-glycolide) encapsulated antibiotic-biphasic calcium phosphate granule complex and additive antibiotic powder in gypsum binder. In this study, the cement was studied further to evaluate its in vitro biological properties (cytocompatibility, platelet activation), anti-infective, and bone regenerative potential in comparison to poly(methyl methacrylate) (PMMA) cement and parenteral therapy in 43 patients (age 5-57years) with chronic MRSA osteomyelitis by analyzing the results of histopathology, radiographs, magnetic resonance imaging, scanning electron microscopy, and serum drug concentrations for 1year. The composite cement showed superior cytocompatibility and coagulant activity compared to PMMA cement. Moreover, the results of different postoperative clinical and radiological examinations also proved the supremacy of composite cement over the other treatment modalities in terms of success rate, faster sepsis control and bone regeneration. Low serum antibiotic concentrations and normal serum calcium levels indicate that the calcium-rich composite cement is safe for application in human. Therefore, we conclude that the composite bone cement is a promising candidate for the treatment of chronic osteomyelitis.

Fabrication of interconnected porous Ag substituted octacalcium phosphate blocks based on a dissolution-precipitation reaction

Here, we introduce Ag substituted octacalcium phosphate (OCP-Ag) blocks with interconnected porous structure and sufficient mechanical strength as bone substitute (i.e., foam). We employed a two-step process for fabrication, which includes a setting reaction for acidic calcium phosphate granules using an acidic phosphate solution and a phase conversion process via dissolution-precipitation method in cocktail ((NH4)2HPO4-NH4NO3-NaNO3-AgNO3) solutions. The Ag contents in the fabricated OCP-Ag foams were 0.08–0.15 at%, which were sufficient in exhibiting contact antibacterial ability. The mechanical strength and porosity of the OCP-Ag foams were about 0.5 MPa and 70%, respectively. These values were sufficient for the application of the OCP-Ag foams as bone substitute.Graphical abstract

Bilateral double site (calvarial and mandibular) critical-size bone defect model in rabbits for evaluation of a craniofacial tissue engineering constructs.

Most existing preclinical models for evaluating the biosafety and bone-regeneration efficacy of innovative bone substitute materials (BSMs) or tissue engineering (TE) constructs only consisted of a single-site defect and the anatomical locations of defect varied drastically. While the compelling evidence showed that the bone healing pattern is location-dependent, owing to developmental, structural, and functional differences of anatomical locations, this is particularly true for the craniofacial region. Taking this into account, the bone healing efficiency of a BSM shown at one anatomical defect location cannot ensure the same impact at another. This prompted us to develop, for the first time, a model of bilateral critical-sized defect (CSD) at two distinctly different locations (non-load-bearing parietal calvaria and load-bearing mandibular body) co-existing in one rabbit to reduce the number of animals needed and avoid the influence of interindividual variability and evaluation bias on comparisons.24 healthy adult male New Zealand White rabbits were randomly assigned to a group, either control, autograft (considered the “gold standard”) or a clinically relevant BSM (biphasic calcium phosphate granules) (BCPg, Mastergraft®, Medronics). The full-thickness cylindrical calvarial defect (ø10 mm) on frontoparietal region and mandibular composite defect (ø11 mm) on the body of the mandible were created bilaterally using low-speed drilling with saline irrigation. The defect on one side was filled with autograft debris or BCPg, and the other side was no graft (empty). Following the euthanasia of animals at the predetermined intervals (4w and 12w), the defect zones were examined macroscopically and then sampled and processed for microcomputed tomography (microCT) and histological analysis.All surgeries went uneventfully, and all rabbits recovered slowly but steadily. No symptoms of infection or inflammation associated with the defect were observed during the experiment. At 4w and 12w, macroscopic views of all defect sites were clean without any signs of necrosis or abscess, and no intraoral communication was found. The analysis of microCT and histological findings showed the non-healing nature of the empty defect, thereby both calvaria and mandible CSDs can be validated. The study of the application of BCPg in this defect model highlighted good osteointegration and excellent osteoconductive properties but compromised the osteoinductive properties of this material (compared with autograft).To conclude, this novel double-site CSD model holds great promise in the application for preclinical evaluation of BSMs, TE construct, etc. With a reduced number of animals in use, and lower interindividual variability and evaluation bias for comparisons.

Phosphorus recovery from pig manure: Dissolution of struvite and formation of calcium phosphate granules during anaerobic digestion with calcium addition

Phosphorus is an essential but finite and scarce element, which is used extensively in pig farming. For recovering phosphorus from pig manure, we hypothesize that calcium addition during anaerobic treatment can trigger calcium phosphate granulation and enable efficient phosphorus recovery. In this study, we tested the recovery of phosphorus from pig manure by adding calcium during anaerobic treatment. Size–separated (<200 µm) pig manure was treated in two identical up-flow anaerobic sludge blanket reactors for 456 days. Calcium was supplied to one of the reactors to study calcium phosphate granulation.Calcium addition showed a positive effect on treatment stability and efficiency, increasing chemical oxygen demand and phosphorus removal. Phosphorus removal increased from 60% without calcium addition to 74% with calcium addition. The addition of calcium increased the phosphorus concentration in significantly larger particles, calcium phosphate granules, (79% >0.4 mm compared with 11% without calcium addition) in the reactor with calcium addition, which enables separation and a transport reduction of 85% for the recovered phosphorus. The main phosphorus phase in the reactor sludge bed changed from struvite to calcium phosphate with calcium addition. The Mg:P molar ratio was 0.09 with calcium addition and 0.88 without calcium addition in particles >2.5 mm. Thus, calcium addition enhances the treatment of pig manure and enables phosphate recovery as calcium phosphate granules.

Biologically Relevant In Vitro 3D-Model to Study Bone Regeneration Potential of Human Adipose Stem Cells.

Standard cell cultures may not predict the proliferation and differentiation potential of human mesenchymal stromal cells (MSCs) after seeding on a scaffold and implanting this construct in a bone defect. We aimed to develop a more biologically relevant in vitro 3D-model for preclinical studies on the bone regeneration potential of MSCs. Human adipose tissue-derived mesenchymal stromal cells (hASCs; five donors) were seeded on biphasic calcium phosphate (BCP) granules and cultured under hypoxia (1% O2) for 14 days with pro-inflammatory TNFα, IL4, IL6, and IL17F (10 mg/mL each) added during the first three days, simulating the early stages of repair (bone construct model). Alternatively, hASCs were cultured on plastic, under 20% O2 and without cytokines for 14 days (standard cell culture). After two days, the bone construct model decreased total DNA (3.9-fold), COL1 (9.8-fold), and RUNX2 expression (19.6-fold) and metabolic activity (4.6-fold), but increased VEGF165 expression (38.6-fold) in hASCs compared to standard cultures. After seven days, the bone construct model decreased RUNX2 expression (64-fold) and metabolic activity (2.3-fold), but increased VEGF165 (54.5-fold) and KI67 expression (5.7-fold) in hASCs compared to standard cultures. The effect of the bone construct model on hASC proliferation and metabolic activity could be largely mimicked by culturing on BCP alone (20% O2, no cytokines). The effect of the bone construct model on VEGF165 expression could be mimicked by culturing hASCs under hypoxia alone (plastic, no cytokines). In conclusion, we developed a new, biologically relevant in vitro 3D-model to study the bone regeneration potential of MSCs. Our model is likely more suitable for the screening of novel factors to enhance bone regeneration than standard cell cultures.

Donor variability alters differentiation and mechanical cohesion of tissue-engineered constructs with human endothelial/MSC co-culture.

To move towards clinical applications, tissue engineering (TE) should be validated with human primary cells and offer easy connection to the native vascularisation. Based on a sheet-like bone substitute developed previously, we investigated a mesenchymal stem cells/endothelial cells (MSCs/ECs) coculture to enhance pre-vascularisation. Using MSCs from six independent donors whose differentiation potential was assessed towards two lineages, we focused on donor variability and cell crosstalk regarding bone differentiation. Coculture was performed on calcium phosphate granules in a specific chamber during 1 month. MSCs were seeded first then ECs were added after 2 weeks, with respective monocultures as control groups. Cell viability and organisation (fluorescence, electronic microscopy), differentiation (ALP staining/activity, RT-qPCR) and mechanical cohesion were analysed. Adaptation of the protocol to coculture was validated (high cell viability and proliferation). Activity and differentiation showed strong trends towards synergistic effects between cell types. MSCs reached early mineralisation stage of maturation. The delayed addition of ECs allowed for their attachment on developed MSCs' matrix. The main impact of donor variability could be here the lack of cell proliferation potential with some donors, leading to low differentiation and mechanical cohesion and therefore absence of sheet-like shape successfully obtained with others. We suggest therefore adapting protocols to cell proliferation potentials from one batch of cells to the other in a patient-specific approach.

Impact of feedwater protein contents on calcium phosphate mineralization in anaerobic digesters

Calcium phosphate (CaP) mineralization and accumulation in anaerobic digesters represent an attractive approach to recovery phosphorus (P) from wastewater. Previous studies demonstrated that under certain anaerobic digestion conditions, favorable localized environment can be developed to facilitate CaP granule growth. However, factors that trigger the CaP nucleation in anaerobic reactors have not been elucidated. In this study, to examine the driving forces in the microenvironment for the CaP nucleation, two laboratory upflow anaerobic sludge blanket (UASB) reactors fed with two types of synthetic wastewater were operated under mesophilic conditions. The feed of one reactor (G reactor) had glucose as the sole carbon source, and the feed of the other reactor (G+B reactor) had glucose (60% chemical oxygen demand (COD)) and bovine serum albumin (BSA, 40% COD) as the combined carbon sources. The results showed that P and Ca removals were only observed in the G+B reactor. The main difference between the two reactors was the elevated pH in the G+B reactor, which may be attributed to the degradation of amino acids. The elevated pH caused the deprotonation of the negatively charged functional groups in the sludge, creating available active surfaces for Ca2+ complexation. The high availability of OH- and the enriched Ca in the G+B reactor built a favorable microenvironment to overcome the activation energy barriers hindering the CaP nucleation. The stabilized CaP mineralization largely depended on the well-established microbial community, where the efficient hydrogenotrophic methanogens and syntrophic acetogens may maintain a stabilized pH environment to prevent the dissociation of CaP minerals.