Injectable Enzymatically Hardened Calcium Phosphate Biocement

(1) Background: The preparation and characterization of novel fully injectable enzymatically hardened tetracalcium phosphate/monetite cements (CXI cements) using phytic acid/phytase (PHYT/F3P) hardening liquid with a small addition of polyacrylic acid/carboxymethyl cellulose anionic polyelectrolyte (PAA/CMC) and enhanced bioactivity. (2) Methods: Composite cements were prepared by mixing of calcium phosphate powder mixture with hardening liquid containing anionic polyelectrolyte. Phase and microstructural analysis, compressive strength, release of ions and in vitro testing were used for the evaluation of cement properties. (3) Results: The simple possibility to control the setting time of self-setting CXI cements was shown (7–28 min) by the change in P/L ratio or PHYT/F3P reaction time. The wet compressive strength of cements (up to 15 MPa) was close to cancellous bone. The increase in PAA content to 1 wt% caused refinement and change in the morphology of hydroxyapatite particles. Cement pastes had a high resistance to wash-out in a short time after cement mixing. The noncytotoxic character of CX cement extracts was verified. Moreover, PHYT supported the formation of Ca deposits, and the additional synergistic effect of PAA and CMC on enhanced ALP activity was found, along with the strong up-regulation of osteogenic gene expressions for osteopontin, osteocalcin and IGF1 growth factor evaluated by the RT-qPCR analysis in osteogenic αMEM 50% CXI extracts. (4) Conclusions: The fully injectable composite calcium phosphate bicements with anionic polyelectrolyte addition showed good mechanical and physico-chemical properties and enhanced osteogenic bioactivity which is a promising assumption for their application in bone defect regeneration

Novel Biocement/Honey Composites for Bone Regenerative Medicine

New biocements based on a powdered mixture of calcium phosphate/monetite (TTCPM) modified with the addition of honey were prepared by mixing the powder and honey liquid components at a non-cytotoxic concentration of honey (up to 10% (w/v)). The setting process of the cements was not affected by the addition of honey, and the setting time of ~4 min corresponded to the fast setting calcium phosphate cements (CPCs). The cement powder mixture was completely transformed into calcium-deficient nanohydroxyapatite after 24 h of hardening in a simulated body fluid, and the columnar growth of long, needle-like nanohydroxyapatite particles around the original calcium phosphate particles was observed in the honey cements. The compressive strength of the honey cements was reduced with the content of honey in the cement. Comparable antibacterial activities were found for the cements with honey solutions on Escherichia coli, but very low antibacterial activities were found for Staphylococcus aureus for all the cements. The enhanced antioxidant inhibitory activity of the composite extracts was verified. In vitro cytotoxicity testing verified the non-cytotoxic nature of the honey cement extracts, and the addition of honey promoted alkaline phosphatase activity, calcium deposit production, and the upregulation of osteogenic genes (osteopontin, osteocalcin, and osteonectin) by mesenchymal stem cells, demonstrating the positive synergistic effect of honey and CPCs on the bioactivity of cements that could be promising therapeutic candidates for the repair of bone defects

The Influence of Nanosilica on Properties of Cement Based on Tetracalcium Phosphate/Monetite Mixture with Addition of Magnesium Pyrophoshate

The effect of nanosilica on the microstructure setting process of tetracalcium phosphate/nanomonetite calcium phosphate cement mixture (CPC) with the addition of 5 wt% of magnesium pyrophosphate (assigned as CT5MP) and osteogenic differentiation of mesenchymal stem cells cultured in cement extracts were studied. A more compact microstructure was observed in CT5MP cement with 0.5 wt% addition of nanosilica (CT5MP1Si) due to the synergistic effect of Mg2P2O7 particles, which strengthened the cement matrix and nanosilica, which supported gradual growth and recrystallization of HAP particles to form compact agglomerates. The addition of 0.5 wt% of nanosilica to CT5MP cement caused an increase in CS from 18 to 24 MPa while the setting time increased almost twofold. It was verified that adding nanosilica to CPC cement, even in a low amount (0.5 and 1 wt% of nanosilica), positively affected the injectability of cement pastes and differentiation of cells with upregulation of osteogenic markers in cells cultured in cement extracts. Results revealed appropriate properties of these types of cement for filling bone defects

Characterization of Tetracalcium Phosphate/Monetite Biocement Modified by Magnesium Pyrophosphate

Magnesium pyrophosphate modified tetracalcium phosphate/monetite cement mixtures (MgTTCPM) were prepared by simple mechanical homogenization of compounds in a ball mill. The MgP2O7 was chosen due to the suitable setting properties of the final cements, in contrast to cements with the addition of amorphous (Ca, Mg) CO3 or newberite, which significantly extended the setting time even in small amounts (corresponding ~to 1 wt% of Mg in final cements). The results showed the gradual dissolution of the same amount of Mg2P2O7 phase, regardless of its content in the cement mixtures, and the refinement of formed HAP nanoparticles, which were joined into weakly and mutually bound spherical agglomerates. The compressive strength of composite cements was reduced to 14 MPa and the setting time was 5–10 min depending on the composition. Cytotoxicity of cements or their extracts was not detected and increased proliferative activity of mesenchymal stem cells with upregulation of osteopontin and osteonectin genes was verified in cells cultured for 7 and 15 days in cement extracts. The above facts, including insignificant changes in the pH of simulated body fluid solution and mechanical strength close to cancellous bone, indicate that MgTTCPM cement mixtures could be suitable biomaterials for use in the treatment of bone defects

Characterization of Properties, In Vitro and In Vivo Evaluation of Calcium Phosphate/Amino Acid Cements for Treatment of Osteochondral Defects

Novel calcium phosphate cements containing a mixture of four amino acids, glycine, proline, hydroxyproline and either lysine or arginine (CAL, CAK) were characterized and used for treatment of artificial osteochondral defects in knee. It was hypothesized that an enhanced concentration of extracellular collagen amino acids (in complex mixture), in connection with bone cement in defect sites, would support the healing of osteochondral defects with successful formation of hyaline cartilage and subchondral bone. Calcium phosphate cement mixtures were prepared by in situ reaction in a planetary ball mill at aseptic conditions and characterized. It was verified that about 30–60% of amino acids remained adsorbed on hydroxyapatite particles in cements and the addition of amino acids caused around 60% reduction in compressive strength and refinement of hydroxyapatite particles in their microstructure. The significant over-expression of osteogenic genes after the culture of osteoblasts was demonstrated in the cement extracts containing lysine and compared with other cements. The cement pastes were inserted into artificial osteochondral defects in the medial femoral condyle of pigs and, after 3 months post-surgery, tissues were analyzed macroscopically, histologically, immunohistochemically using MRI and X-ray methods. Analysis clearly showed the excellent healing process of artificial osteochondral defects in pigs after treatment with CAL and CAK cements without any inflammation, as well as formation of subchondral bone and hyaline cartilage morphologically and structurally identical to the original tissues. Good integration of the hyaline neocartilage with the surrounding tissue, as well as perfect interconnection between the neocartilage and new subchondral bone tissue, was demonstrated. Tissues were stable after 12 months’ healing

In Vivo Study of Osteochondral Defect Regeneration Using Innovative Composite Calcium Phosphate Biocement in a Sheep Model

This study aimed to clarify the therapeutic effect and regenerative potential of the novel, amino acids-enriched acellular biocement (CAL) based on calcium phosphate on osteochondral defects in sheep. Eighteen sheep were divided into three groups, the treated group (osteochondral defects filled with a CAL biomaterial), the treated group with a biocement without amino acids (C cement), and the untreated group (spontaneous healing). Cartilages of all three groups were compared with natural cartilage (negative control). After six months, sheep were evaluated by gross appearance, histological staining, immunohistochemical staining, histological scores, X-ray, micro-CT, and MRI. Treatment of osteochondral defects by CAL resulted in efficient articular cartilage regeneration, with a predominant structural and histological characteristic of hyaline cartilage, contrary to fibrocartilage, fibrous tissue or disordered mixed tissue on untreated defect (p < 0.001, modified O’Driscoll score). MRI results of treated defects showed well-integrated and regenerated cartilage with similar signal intensity, regularity of the articular surface, and cartilage thickness with respect to adjacent native cartilage. We have demonstrated that the use of new biocement represents an effective solution for the successful treatment of osteochondral defects in a sheep animal model, can induce an endogenous regeneration of cartilage in situ, and provides several benefits for the design of future therapies supporting osteochondral defect healing