Kunio Ishikawa

Zinc-releasing calcium phosphate for stimulating bone formation ☆

Zinc-containing tricalcium phosphate (ZnTCP) is biocompatible and bioactive, and functions as an effective zinc carrier. ZnTCP contains a maximum of 12 mol% of zinc. ZnTCP ceramics and composite ceramics of ZnTCP and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 were prepared by sintering at 1100 °C. ZnTCP/HAP continued to release zinc for more than 50 days in 0.9 wt.% sodium chloride solution. ZnTCP/HAP with a zinc content of 1.2 wt.% significantly increased osteoblastic MC3T3-E1 cell proliferation and alkaline phosphatase activity of rat stromal cells in vitro. ZnTCP/HAP with a zinc content of 0.316 wt.% increased bone formation by 51% (n=6; p=0.0509) 4 weeks after implantation in rabbit compared to the control without zinc. A zinc concentration within a noncytotoxic level of a solution does not completely block the crystal growth of apatite in the solution. When ZnTCP is added to calcium phosphate cement, the ZnTCP showed neither inhibitory nor promoting effect on the setting ability of the cement. All these findings suggest that the zinc-containing tricalcium phosphate is a biomaterial that has a pharmaceutical effect of promoting bone formation.

Non-decay type fast-setting calcium phosphate cement: composite with sodium alginate.

Non-decay type fast-setting calcium phosphate cement (nd-FSCPC) was prepared by introducing sodium alginate (0-2.0 wt%) into the liquid phase of FSCPC. nd-FSCPC was stable even when the cement paste was immersed in distilled water immediately after mixing, whereas conventional FSCPC (c-FSCPC) decayed completely within 1 min upon immersion. The setting time of the cement, approximately 5 min, was not dependent on the presence of sodium alginate. In contrast, the introduction of sodium alginate into conventional CPC, i.e. CPC without neutral phosphate in the liquid phase, resulted in no setting when the amount of sodium alginate introduced was more than 1 wt%. Powder X-ray diffraction analysis revealed no significant difference for the conversion of cement to apatite for any concentrations of sodium alginate studied (0-2.0 wt%). The mechanical strength of the cement increased rapidly with the addition of sodium alginate up to 0.8 wt% when the cement paste was immersed and kept in distilled water at 37 degrees C, whereas further addition of sodium alginate decreased the mechanical strength. The results obtained in this investigation, taken together with sodium alginates known excellent biocompatibility and absorption behaviour, indicate that the use of sodium alginate composite FSCPC as nd-FSCPC should be of value in orthodontics and oral and maxillofacial surgery where the cement is exposed to blood.

Determination of the Ca/P ratio in calcium- deficient hydroxyapatite using X-ray diffraction analysis

The determination of the calcium to phosphate ratio (Ca/P) in Ca-deficient hydroxyapatite [d-HAP; Ca10−x(HPO4)x(PO4)6−x(OH)2−x] using X-ray diffraction analysis (XRD) is reported. At temperatures above 700°C HPO42- groups are transformed to PO43- groups, thereby producing β-tricalcium phosphate [β-TCP; Ca3(PO4)2]. Thus, the deviation from stoichiometry, x, can be calculated from the mass fraction of β-TCP, which in turn can be determined from quantitative XRD analyses. In this study d-HAP powders with various Ca/P ratios were prepared following several procedures. It is shown that the Ca/P ratio determined by quantitative XRD correlates well with that measured by chemical analyses.

Formation of hydroxyapatite in new calcium phosphate cements.

Tetracalcium phosphate (TTCP) has been shown previously to be an essential component of self-setting calcium phosphate cements that form hydroxyapatite (HA) as the only end-product. We report herein on a new self-setting calcium phosphate cement that does not contain TTCP. These cements consist of dicalcium phosphate anhydrous (DCPA), dicalcium phosphate dihydrate (DCPD), alpha-tricalcium phosphate, or amorphous calcium phosphate and, as an additional source of calcium, calcium hydroxide or calcium carbonate. These cements require the use of a phosphate (0.2 moll(-1) or higher) solution or a high pH solution as the cement liquid. The cements harden in relatively short time (5-30 min) and form HA as the dominant end-product in 24 h. The diametral tensile strengths of the 24-h samples are in the range of 0.2 to 7.5 MPa. Results from X-ray diffraction studies suggest that the cement setting is caused by rapid HA formation induced by the high phosphate concentration of the cement liquid. Because DCPA and DCPD are highly soluble at pH values above 12.7, which is the pK3 of phosphoric acid, high phosphate concentration in the slurry solution was also attainable by using a highly alkaline solution as the cement liquid. The physicochemical properties of these cements are comparable to those of TTCP-containing cements, and the new cements may be expected to have in vivo characteristics similar to those of TTCP-containing cements as well.

Properties and mechanisms of fast-setting calcium phosphate cements

The setting time of a calcium phosphate cement consisting of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) was reduced from 30 to 5 min by use of a cement liquid that contained a phosphate concentration of 0.25 mol/l or higher. The diametral tensile strength and conversion of the cement ingredients to hydroxyapatite (OHAp) during the first 3 h were also significantly increased by the phosphate. However, the phosphate produced no significant effects on the properties of the 24-h cement samples. Results from additional experiments in a slurry system verified that the high phosphate concentration in the solution accelerated the formation of OHAp in the TTCP + DCPA system, and this reaction could explain the fast-setting properties of the cements.

In vivo setting behaviour of fast-setting calcium phosphate cement

The in vivo setting behaviour of fast-setting calcium phosphate cement (FSCPC) between femoral muscles of the rat was investigated to evaluate the possible value of FSCPC for medical and dental application. Conventional CPC (c-CPC) and FSCPC were implanted between femoral muscles, and various aspects of the setting behaviour such as setting time, mechanical strength and conversion ratio of cement into hydroxyapatite (HAP: Ca10(PO4)6(OH)2) were measured by the Vicat needle method, diametral tensile strength (DTS) measurement, and quantitative powder X-ray diffraction (XRD) analysis, respectively. The setting time of FSCPC in vivo was 5-7 min, in contrast to 48 min for c-CPC. As a result of its fast setting, set specimens of FSCPC showed higher mechanical strength from the initial stage than c-CPC. Higher DTS values were observed in FSCPC than c-CPC implanted after 24 h. Powder XRD analysis revealed faster conversion of FSCPC than c-CPC into HAP, which was responsible both for the faster setting and higher mechanical strength from the initial stage. We concluded, therefore, that FSCPC may be used for a wide range of clinical applications, i.e. fields where fast setting is required such as orthopaedic, plastic and reconstructive, and oral and maxillofacial surgery.

Histological and compositional evaluations of three types of calcium phosphate cements when implanted in subcutaneous tissue immediately after mixing

To evaluate the soft tissue response of calcium phosphate cement (CPC), consisting of an equimolar mixture of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) under conditions close to those encountered in actual surgical procedures, we implanted three types of CPC [conventional CPC (c-CPC), fast-setting CPC (FSCPC), and antiwashout type FSCPC (aw-FSCPC; formerly called nondecay type FSCPC or nd-FSCPC)] subcutaneously in the abdomens of rats immediately (1 min) after mixing. At 1 week after surgery, histological examination and compositional analysis were performed using light microscopy and powder X-ray diffraction (XRD), respectively. The implanted c-CPC was crumbled completely, whereas FSCPC and aw-FSCPC retained their shape. Large vesicles containing copious inflammatory effusion were subcutaneously formed around the c-CPC. Histologically, many foreign-body giant cells were collected around the c-CPC, and moderate inflammatory cell infiltration was observed at 1 week after surgery. In contrast, the FSCPC and aw-FSCPC were covered with a thin layer of granulation tissue that included few giant cells and presented slight inflammatory cell infiltration, and no effusion was observed. The XRD analysis of the c-CPC revealed the presence of some unreacted DCPA even 1 week after implantation, whereas almost no DCPA was found in the FSCPC or aw-FSCPC. In conclusion, it was found that CPC does not always show excellent tissue response. When c-CPC is implanted subcutaneously in rats immediately after mixing, it fails to set and causes a severe inflammatory response. Therefore, the type of CPC should be chosen according to the clinical particulars. CPC should be used in a manner that assures its setting reaction. We recommend the use of FSCPC and aw-FSCPC for surgical applications, such as orthopedics, plastic and reconstructive surgery, and oral and maxillofacial surgery, where the cement might otherwise crumble due to the pressure before setting.

Non-decay type fast-setting calcium phosphate cement: Hydroxyapatite putty containing an increased amount of sodium alginate

A hydroxyapatite [(HAP) Ca10(PO4)6(OH)2] putty that behaves like a putty or self-curing resin was made by increasing the amount of sodium alginate in non-decay type fast-setting calcium phosphate cement (nd-FSCPC). nd-FSCPC became viscous as the sodium alginate concentration was increased. The best handling properties were obtained when nd-FSCPC contained 8% sodium alginate in its liquid phase. When a 2-kg glass plate was placed on the paste, HAP putty spread to form an area three times that of FSCPC paste. Thus, HAP putty is expected to be easier to use than FSCPC in the filling of bone defects. HAP putty did not decay; in fact, it set within approximately 20 min when immersed in distilled water immediately after mixing. The wet diametral tensile strength value of HAP putty was approximately 12 MPa after 24 h, the same as that for nd-FSCPC containing 0.5% sodium alginate in its liquid phase, or FSCPC that is free from sodium alginate. The elements constituting set HAP putty were examined using powder X-ray diffraction and found to be predominantly apatitic minerals after 24 h. Since the handling properties of a putty or self-curing resin-like cement are very useful in certain surgical procedures, HAP putty made by increasing the sodium alginate concentration in nd-FSCPC is potentially a valuable new biomaterial for use in plastic and reconstructive surgery, as well as oral and maxillofacial surgery.

Basic properties of calcium phosphate cement containing atelocollagen in its liquid or powder phases

The basic properties of calcium phosphate cement (CPC) containing atelocollagen, the main component of the organic substrate in bone, were studied in an initial evaluation for the fabrication of modified CPC. The setting time of conventional CPC (c-CPC) was prolonged to over 100 min when c-CPC contained 1% or more atelocollagen. The diametral tensile strength (DTS) of c-CPC decreased linearly with the collagen content, descending to below the detection limit when the c-CPC contained 3% or more atelocollagen. Therefore, use of c-CPC as the base cement seems inappropriate for the fabrication of atelocollagen-containing CPC. In contrast, the cement set at 9-34 min when fast-setting CPC (FSCPC) was used as the base cement and contained 1-5% atelocollagen, respectively. Although addition of atelocollagen resulted in the decrease of DTS of the set mass, the DTS was approximately the same, 6-8 MPa, at contents of atelocollagen between 1% and 5%. When atelocollagen was added to FSCPC, the handling property was improved significantly. The paste also became more adhesive with increase in atelocollagen content. These properties are desirable for its use in surgical procedures since, for example, bony defects can be filled easily and without a space interposed between the bone and cement paste. Although there are some disadvantages for the addition of atelocollagen to CPC, it can be accepted as long as FSCPC was used as the base cement. We conclude that further evaluations of the effects of atelocollagen, such as biocompatibility, bone synthesis, and bone replacement behaviour should be done, using FSCPC as the base cement.

Effects of added antibiotics on the basic properties of anti-washout-type fast-setting calcium phosphate cement.

The effect of added antibiotics on the basic properties of anti-washout-type fast-setting calcium phosphate cement (aw-FSCPC) was investigated in a preliminary evaluation of aw-FSCPC containing drugs. Flomoxef sodium was employed as the antibiotic and was incorporated into the powder-phase aw-FSCPC at up to 10%. The setting time, consistency, wet diametral tensile strength (DTS) value, and porosity were measured for aw-FSCPC containing various amounts of flomoxef sodium. X-ray diffraction (XRD) analysis was also conducted for the identification of products. To evaluate the drug-release profile, set aw-FSCPC was immersed in saline and the released flomoxef sodium was determined at regular intervals. The spread area of the cement paste as an index of consistency of the cement increased progressively with the addition of flomoxef sodium, and it doubled when the aw-FSCPC contained 8% flomoxef sodium. In contrast, the wet DTS value decreased with increase in flomoxef sodium content. Bulk density measurement and scanning electron microscopic observation revealed that the set mass was more porous with the amount of flomoxef sodium contained in the aw-FSCPC. The XRD analysis revealed that formation of hydroxyapatite (HAP) from aw-FSCPC was reduced even after 24 h, when the aw-FSCPC contained flomoxef sodium at > or = 6%. Therefore, the decrease of wet DTS value was thought to be partly the result of the increased porosity and inhibition of HAP formation in aw-FSCPC containing large amounts of flomoxef sodium. The flomoxef sodium release from aw-FSCPC showed the typical profile observed in a skeleton-type drug delivery system (DDS). The rate of drug release from aw-FSCPC can be controlled by changing the concentration of sodium alginate. Although flomoxef sodium addition has certain disadvantageous effects on the basic properties of aw-FSCPC, we conclude that aw-FSCPC is a good candidate for potential use as a DDS carrier that may be useful in surgical operations.