Summary of the invention
The purpose of this invention is to provide a kind of biogenic property and fluoridize the method for preparing of hydroxyapatite bone alternate material, this method as synthetic raw material, is passed through the pig femur network that had both had suitable cell growth in the bone alternate material that is obtained behind physical treatment and the chemical fluoridize with the pig femur; And contain an amount of fluorine; Can satisfy the damaged reparation of bone clinically, or be applied to bone tissue engineer, have the national conditions of meeting again as cytoskeleton; Wide material sources, lower-price characteristic.
The object of the invention is realized through following technical scheme: a kind of biogenic property is fluoridized the method for preparing of hydroxyapatite bone alternate material, may further comprise the steps:
(1) draw materials: the femoral inferior segment spongy bone to become pig is a raw material, is sawn into the bone piece, drying for standby;
(2) sintering: to the bone piece sintering that heats up, temperature to be sintered rises to 700 ~ 900 ℃ under air atmosphere, and sintering is 1 ~ 2 hour under this temperature range, cleans up after the room temperature cooling then, and drying obtains Os Sus domestica hydroxyapatite (PHA);
(3) soak: the Os Sus domestica hydroxyapatite soaked in fluorine-containing solution 12 ~ 24 hours, took out drying;
(4) reacting by heating: will pass through Os Sus domestica hydroxyapatite after step (3) is handled and place and be warming up to 700 ~ 800 ℃ under the aerobic environment; And this temperature range internal heating 2 ~ 3 hours, cooling afterwash, drying; Pulverize obtains biogenic property and fluoridizes hydroxyapatite (FPHA) bone alternate material.
In the common animals, the similarity the highest (referring to table 1) of Os Sus domestica and people's bone.And the inventor discovers, the femoral inferior segment spongy bone of pig has the micropore that is suitable for cell growth, and simultaneously, its macroscopic mesh-like structure also helps flowing and perfusion of blood, and in preparation, spongy bone is beneficial to shaping, clinically also easy operating.Thereby the femoral inferior segment spongy bone that can adopt pig is as raw material.
Table 1: the similarity of animals and human beings bone
|
Os Canitis |
Os Caprae seu Ovis |
Os Sus domestica |
Os Leporis seu Oryctolagi |
Macrostructure |
++ |
+++ |
++ |
+ |
Microstructure |
++ |
+ |
++ |
+ |
Bone component |
+++ |
- |
+++ |
++ |
Bone is rebuild |
++ |
++ |
+++ |
+ |
Annotate :+low similar, ++ moderate is similar, +++highly similar
But because the organic matter that biological bone contains can cause the intravital rejection of people, something must be done to when therefore using clinically removes fully.Find that after deliberation spongy bone is when 700 ℃ of left and right sides sintering, the organic matter in the bone almost can be removed fully; And be higher than 1000 ℃ high temperature sintering, just along with the difference of sintering time, decomposition in various degree takes place in HA; (β-TCP), its bone conductibility is weaker than HA, and absorption rate is fast than HA to produce tricalcium phosphate; Can influence the bone grafting effect, therefore, the sintering temperature of Os Sus domestica is controlled at 700 ~ 900 ℃.And Os Sus domestica HA mixes about 700 ~ 900 ℃ and does not almost have organic residual, no β-TCP and other unformed apatite among the FPHA that the fluorine sintering obtains after 1 ~ 2 hour; If but continue heating; Or temperature is too high; Then can cause the carbonate in the carbonated hydroxyapatite to decompose, temperature is crossed low or the too short fluorion of sintering time then is difficult to chemical mode and combines with PHA, can not reach the alternate purpose of fluorine.Therefore, in order to preserve this carbonate as far as possible, make it to preserve the advantage of biogenic property HA, the temperature of selecting 700 ~ 800 ℃ among the present invention is as the treatment temperature of step (4) reacting by heating.Both preserve carbonate, guaranteed the doping of fluorine again.
The volume size of the bone piece described in the step according to the invention (1) is 1 * 1 * 0.5cm
3, be beneficial to make in the subsequent step bone piece to be heated evenly, so that Organic substance is removed fully, fluorion is evenly implanted.
In the step according to the invention (1), the bone piece is at room temperature dry to get final product, and can not do other special handlings.As for improving the exsiccant efficient of bone piece, can adopt the mode of strengthening circulation of air to carry out drying.
The used in amounts of fluorine-containing solution satisfies the absorption requirement of PHA in the step according to the invention (3), normally adopts excessive Fluorinse to soak, and for example every 100ml Fluorinse soaks 1g PHA.The bone piece itself has loose structure, and Fluorinse did not have the bone piece can reach the even contact immersion.
Fluorine-containing solution in the step according to the invention (3) adopts sodium fluoride (NaF) solution, and the concentration of described Fluorinse is 0.25 ~ 1mol/L, is preferably 0.25 ~ 0.5mol/L.
Temperature is to rise to 700 ~ 900 ℃ from room temperature in the intensification sintering in the step according to the invention (2); The heating rate of temperature generally is advisable to be no more than 10 ℃/min; Cause Organic substance moment burning easily because heating rate is too fast, local temperature is too high, and the intermedium crystalline phase that causes being obtained changes.
Likewise, the heating rate of the heating-up temperature in the step according to the invention (3) generally is advisable to be no more than 10 ℃/min, avoids that heating rate is too fast to cause the burning of Organic substance moment to cause local temperature too high, prevents that the intermedium crystalline phase that is obtained from changing.
The drying that relates in step according to the invention (3) and (4) is being carried out below 80 ℃.
In step according to the invention (1) ~ (4), after drying, all need semifinished or finished goods be preserved at a distance from wet, avoid product to make moist, influence the result of use of semi-finished product at the treatment effect and the finished product of each step.
The present invention compared with prior art has following beneficial effect:
(1) the present invention is main synthetic raw material with Os Sus domestica, and its macrostructure, microstructure aspect are the most similar with people's bone, and the similarity of composition, bone density and people's bone is only second to Os Canitis., F obtains FPHA after substituting.Experiment confirm, this FPHA have kept the biological framework of HA, and are retaining the more intravital favourable trace element of animal; Compare with pure biogenic HA,, have stronger osteoinductive more helping growing into of osteocyte; New osteogenesis better effects if, range of application is more extensive, can better application in the damaged reparation of osseous tissue; The support of bone tissue engineer makes up, implant surface coating etc.
(2) the present invention is raw material with the Os Sus domestica, and is more extensive than Os Bovis seu Bubali, cheap in domestic its source of China; Help reducing the cost of raw material; And need not to take precautions against with Hinduism etc. is religious and conflict and medical-risk that bovine spongiform encephalopathy Os Bovis seu Bubali etc. brings, make application wider of product, simultaneously; Help simplifying preparation technology, thereby can reduce the cost of FPHA greatly.
For example; The Bio-oss that the Switzerland Co., Ltd of the import of using at present produces, its purchase price is 2000 yuan of every grams, and presses the Os Sus domestica of existing market valency purchase; Prepare according to the inventive method; Per 150 yuan Os Sus domestica can make 15g biogenic property FPHA, 100 yuan of its every gram cost less thaies, and selling price also can be far below the selling price of Bio-oss.It is thus clear that use of the present invention can fundamentally reduce patient's treatment cost, have sizable market competitiveness.
(3) to make the alternate FPHA of variable concentrations F, technology is very simple, is easy to realize the production of scale through the concentration of adjustment fluoride aqueous solution in the present invention.
(4) the present invention also can realize the cycling and reutilization of the Os Sus domestica of discarding in the past or only limiting to eat is turned waste into wealth.
The specific embodiment
Below set forth content of the present invention in further detail through embodiment and experimental example; But following embodiment just is used for content of the present invention is set forth; Rather than restriction; Therefore in implication suitable and any change in the scope, all should think to be included in the scope of claims with claims of the present invention.
Embodiment 1
(1) draws materials: get into pig femoral inferior segment spongy bone, remove most soft tissue, clean, be sawn into about 1 * 1 * 0.5cm of size
3The bone piece, drying at room temperature is subsequent use.
(2) sintering: variations in temperature in the bone piece sintering that in Muffle furnace, in air, heats up, programme-control stove rises to 800 ℃ with the heating rate of 10 ℃/min from room temperature; Kept sintering 2 hours at 800 ℃ then, take out after being cooled to room temperature, washed with de-ionized water once; Remove the residue on bone piece surface, filter 80 ℃ of dryings; Obtain Os Sus domestica hydroxyapatite (PHA), preserve at a distance from wet then.
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.25mol/LNaF solution, let PHA be soaked in the excessive N aF solution, take out PHA behind the immersion 24h; All PHA preserve at a distance from wet in 80 ℃ of dryings.
(4) reacting by heating: will place clean Muffle furnace through the PHA that step (3) is handled; Under aerobic environment, carry out reacting by heating and handle, heating-up temperature is warming up to 700 ℃ with 10 ℃/min of heating rate from room temperature, and PHA keeps heating 3 hours in 700 ℃ of temperature then; Heat treated finishes the back taking-up and cleans 1min repeatedly with deionized water; Be milled into powdery after 80 ℃ of dryings, obtain biogenic property and fluoridize hydroxyapatite (FPHA) bone alternate material, preserve at a distance from wet.
Sample is carried out physicochemical property detect and zoopery, the result is referring to experimental example.
Embodiment 2
Different with embodiment one is:
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.50mol/LNaF solution, let PHA be soaked in the excessive N aF solution, take out PHA behind the immersion 12h; All PHA preserve at a distance from wet in 80 ℃ of dryings.
(4) reacting by heating: will place clean Muffle furnace under aerobic environment, to carry out reacting by heating through the PHA that step (3) is handled and handle; Heating-up temperature is warming up to 750 ℃ with 10 ℃/min of heating rate from room temperature; PHA keeps heating 2.5 hours in 750 ℃ of temperature then, and heat treated finishes the back taking-up and cleans 1min repeatedly with deionized water, is milled into powdery after 80 ℃ of dryings; Obtain biogenic property and fluoridize hydroxyapatite (FPHA) bone alternate material, preserve at a distance from wet.
Sample is carried out physicochemical property detect and zoopery, the result is referring to experimental example.
Embodiment 3
Different with embodiment one is:
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.75mol/LNaF solution, let PHA be soaked in the excessive N aF solution, take out PHA behind the immersion 20h; All PHA preserve at a distance from wet in 80 ℃ of dryings.
(4) reacting by heating: will place clean Muffle furnace under aerobic environment, to carry out reacting by heating through the PHA that step (3) is handled and handle; Heating-up temperature is warming up to 800 ℃ with 10 ℃/min of heating rate from room temperature; PHA keeps heating 2 hours in 800 ℃ of temperature then, and heat treated finishes the back taking-up and cleans 1min repeatedly with deionized water, is milled into powdery after 80 ℃ of dryings; Obtain biogenic property and fluoridize hydroxyapatite (FPHA) bone alternate material, preserve at a distance from wet.
Sample is carried out physicochemical property detect and zoopery, the result is referring to experimental example.
Embodiment 4
Different with embodiment one is:
(3) soak: by the ratio of every 100ml Fluorinse immersion 1gPHA, get 1gPHA and be dipped in 100ml1.00mol/LNaF solution, take out PHA after soaking 18h respectively; All PHA preserve at a distance from wet in 80 ℃ of dryings.
Sample is carried out physicochemical property detect and zoopery, the result is referring to experimental example.
Embodiment 5
(1) draws materials: get into pig femoral inferior segment spongy bone, remove most soft tissue, clean, be sawn into about 1 * 1 * 0.5cm of size
3The bone piece, drying at room temperature is subsequent use.
(2) sintering: variations in temperature in the bone piece sintering that in Muffle furnace, in air, heats up, programme-control stove rises 700 ℃ with the heating rate of 10 ℃/min from room temperature; Kept sintering 2.5 hours at 700 ℃ then, take out after being cooled to room temperature, washed with de-ionized water once; Remove the residue on bone piece surface, filter 80 ℃ of dryings; Obtain Os Sus domestica hydroxyapatite (PHA), preserve at a distance from wet then.
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml 0.25mol/LNaF solution, let PHA be soaked in the excessive N aF solution, take out PHA behind the immersion 24h; All PHA preserve at a distance from wet in 80 ℃ of dryings.
(4) reacting by heating: will place clean Muffle furnace under aerobic environment, to carry out reacting by heating through the PHA that step (3) is handled and handle; Heating-up temperature is warming up to 800 ℃ with 10 ℃/min of heating rate from room temperature; PHA keeps heating 2 hours in 800 ℃ of temperature then, and heat treated finishes the back taking-up and cleans 1min repeatedly with deionized water, is milled into powdery after 80 ℃ of dryings; Obtain biogenic property and fluoridize hydroxyapatite (FPHA) bone alternate material, preserve at a distance from wet.
Embodiment 6
(1) draws materials: get into pig femoral inferior segment spongy bone, remove most soft tissue, clean, be sawn into about 1 * 1 * 0.5cm of size
3The bone piece, drying at room temperature is subsequent use.
(2) sintering: variations in temperature in the bone piece sintering that in Muffle furnace, in air, heats up, programme-control stove rises 900 ℃ with the heating rate of 10 ℃/min from room temperature; Kept sintering 1 hour at 900 ℃ then, take out after being cooled to room temperature, washed with de-ionized water once; Remove the residue on bone piece surface, filter 80 ℃ of dryings; Obtain Os Sus domestica hydroxyapatite (PHA), preserve at a distance from wet then.
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.05mol/LNaF solution, let PHA be soaked in the excessive N aF solution, take out PHA behind the immersion 20h; All PHA preserve at a distance from wet in 80 ℃ of dryings.
(4) reacting by heating: will place clean Muffle furnace under aerobic environment, to carry out reacting by heating through the PHA that step (3) is handled and handle; Heating-up temperature is warming up to 700 ℃ with 10 ℃/min of heating rate from room temperature; PHA keeps heating 3 hours in 700 ℃ of temperature then, and heat treated finishes the back taking-up and cleans 1min repeatedly with deionized water, is milled into powdery after 80 ℃ of dryings; Obtain biogenic property and fluoridize hydroxyapatite (FPHA) bone alternate material, preserve at a distance from wet.
Embodiment 7
(1) draws materials: get into pig femoral inferior segment spongy bone, remove most soft tissue, clean, be sawn into about 1 * 1 * 0.5cm of size
3The bone piece, drying at room temperature is subsequent use.
(2) sintering: variations in temperature in the bone piece sintering that in Muffle furnace, in air, heats up, programme-control stove rises 750 ℃ with the heating rate of 10 ℃/min from room temperature; Kept sintering 2.5 hours at 750 ℃ then, take out after being cooled to room temperature, washed with de-ionized water once; Remove the residue on bone piece surface, filter 80 ℃ of dryings; Obtain Os Sus domestica hydroxyapatite (PHA), preserve at a distance from wet then.
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.04mol/LNaF solution, let PHA be soaked in the excessive N aF solution, take out PHA behind the immersion 24h; All PHA preserve at a distance from wet in 80 ℃ of dryings.
(4) reacting by heating: will place clean Muffle furnace under aerobic environment, to carry out reacting by heating through the PHA that step (3) is handled and handle; Heating-up temperature is warming up to 750 ℃ with 10 ℃/min of heating rate from room temperature; PHA keeps heating 3 hours in 750 ℃ of temperature then, and heat treated finishes the back taking-up and cleans 1min repeatedly with deionized water, and 80 ℃ of dry backs are with being milled into powdery; Obtain biogenic property and fluoridize hydroxyapatite (FPHA) bone alternate material, preserve at a distance from wet.
Experimental example:
The sample that makes with embodiment 1 ~ 4 is an example, respectively it is carried out physicochemical property detection, cell in vitro detection, animal vivo test detection.Experiment component is that PHA group, contrast PHA organize, soak PHA group and FPHA group, and wherein the PHA group is the Os Sus domestica source property hydroxyapatite (PHA) that the present invention passes through the high temperature sintering preparation, and it is consistent with clinical biogenic HA composition commonly used to detect confirmation through experiment; Contrast PHA group is to carry out the PHA that reacting by heating is handled gained by preparing identical method with FPHA after soaking PHA with deionized water, and PHA group and contrast PHA group are all as testing reference with comparative evaluation FPHA physics and chemistry and biology performance; Soak that the PHA group is soaked in Fluorinse for PHA but the meso sample processed of sintering not, wherein contain certain density fluorion, but non-chemically implant, be soaked in the liquid and can separate out loss fast for physical absorption.
One, physicochemical property
PHA and FPHA with embodiment 1 ~ 4 are example, adopt SEM, XPS, and FTIR, XRD, EDS, TEM, micro-solid titration method is carried out the physicochemical property detection to the FPHA finished product of embodiment 1 ~ 4.Experiment component is PHA group, contrast PHA group, soaks PHA group and FPHA group, and the physicochemical property testing result is following:
1. morphology analysis:
Sample with embodiment 2 is an example, and like Fig. 1, the scanning electron microscope shown in 2 and 3 (SEM) testing result, pattern is similar substantially with FPHA for PHA, is loose porous space network; High power lens can be found the crystal grain of FPHA down for long bar-shaped, and form is more regular than PHA, and degree of crystallinity is higher, and the crystal grain of PHA and blank group is lamellar, irregular shape.
2. analysis of components:
According to X-ray microcell energy spectrum analysis (EDS) testing result, PHA and FPHA all mainly are made up of calcium, phosphorus and oxygen element, also contain a certain amount of carbon and Trace Sodium and magnesium.Its difference is, contains a certain amount of fluorine through the FPHA that fluoridizes gained.Composition is as shown in table 2 in detail.
Table 2:EDS half-quantitative detection PHA and FPHA composition
3. crystalline phase analysis:
X-ray diffraction analysis (XRD) analysis result as shown in Figs. 4a and 4b, the X-ray diffraction peak of PHA and FPHA is similar, can be corresponding one by one with JCPDS72-1243 standard control card.Cause its lattice to shrink because fluorine is implanted PHA, degree of crystallinity increases, and the X-ray diffraction peak of experimental group FPHA is slightly to high angle of diffraction drift.
4. functional group analysis:
According to Fourier's infrared spectrum (FTIR) testing result, all contain carbonate group like Fig. 5 a and 5b among PHA and the FPHA, promptly this two be carbonated hydroxyapatite.In addition,, substituted part of hydroxyl, made that the absworption peak of hydroxyl disappears among the FPHA because fluorion is implanted in the PHA lattice.
5. fluorine chemistry state analysis:
The binding energy that shows fluorine among X line electron spectroscopy analysis (XPS) result reduces, and shows that variation has taken place its chemical state, and promptly its residing chemical micro-environment changes, changes in HA the state with calcium, phosphorus, the multiple interaction between atoms of oxygen into by Na-F.Concrete delta data is as shown in table 3.Wherein, C represents artificial synthetic HA; B represents PHA; The contrast PHA of B-heat representative after heat treated, the immersion PHA of 0.50mol/l Fluorinse has been soaked in A (0.50mol/l) representative, and A-heated (0.50mol/l) has represented through the immersion of heat treated the FPHA of 0.50mol/l Fluorinse; The immersion PHA of 1.00mol/l Fluorinse has been soaked in A (1.00mol/l) representative, and A-heated (1.00mol/l) has represented through the immersion of heat treated the FPHA of 1.00mol/l Fluorinse.
Table 3: laboratory sample XPS testing result
Group |
Fluorine element peak value (eV) |
Constituent content (%) |
C |
- |
0 |
B |
- |
0 |
B-heated |
- |
0 |
A(0.50mol/l) |
685.09 |
3.61 |
A-heated(0.50mol/l) |
684.38 |
2.10 |
A(1.00mol/l) |
685.71 |
4.23 |
A-heated(1.00mol/l) |
685.05 |
3.82 |
6. dissolubility
The most situation of bone-grafting material all are to be applied in to need the spatial zone of long term maintenance bone grafting clinically at present, can not absorb or absorb slowly bone-grafting material and be first and select, and when carrying out bone grafting in the aesthstic district of the upper jaw, recommend to use to absorb bone-grafting material slowly.Promptly concerning bone-grafting material, the bone alternate material of low solubility has greater advantages.Adopt the solid titration method to measure the dissolubility of PHA and HA, as shown in Figure 6, the PHA dissolubility is lower than HA, is about 1/4th of HA, explains that the PHA absorption rate can be lower than HA.And having document to confirm, the phosphorus ash stone crystal is replaced the back dissolubility at the OH group by F can further be reduced, so FPHA also has suitable advantage aspect dissolubility.
Visible from above-mentioned physicochemical property experimental result, the PHA sample of embodiment 2 is consistent with clinical biogenic HA composition commonly used, and main component is HA, contains the inorganic constituents that a small amount of natural bone tissue has simultaneously; The FPHA finished product is merely the fluorion chemical replacement and replaces the OH group gained among the PHA, and other material compositions do not change, and have the physicochemical property of fluoridizing hydroxyapatite.And the high temperature sintering condition of embodiment 1 ~ 4 is identical, and the heat treated temperature is close, and the FPHA performance of therefore final gained is close, all has the physicochemical property of fluoridizing hydroxyapatite.Below select the FPHA finished product of embodiment 1 to experimentize, verified the bone formation performance of the FPHA finished product of gained of the present invention.
Two, the release of the sticking of cell, propagation, differentiation and fluorine
Experimental apparatus
Artificial intelligence's tube type resistance furnace SGM6812BK, Luoyang, Henan, China
ZP10A rotary tablet machine Tianjin good fortune unit inscription instrument and equipment company limited, China
GM-2 type cobalt-60 gamma irradiation device Beijing gamma new and high technology company limited, China
Electronic balance ME235S, Sartorius, Germany
Superclean bench Kendrol, Germany
CO2 constant temperature incubator Shel Lab, the U.S.
Axiovert40 inverted microscope Zeiss, Japan
Electric heating constant temperature air dry oven DHG-9420A, Shanghai, China
Constant water bath box Shel Lab, the U.S.
Ultra-pure water cleaning system Millipore, Elix, the U.S.
Enzyme-linked immunosorbent assay instrument Tecan, Australia
Tabletop refrigerated centrifuge Kendro, the U.S.
QUANTA200 environmental scanning electron microscope FEI Company, Holland
Adjustable pipette Eppendrof, Germany
0.22 μ m filter Millipore, the U.S.
Ionic-activity meter L2850765, the Western Regions are dynamo-electric, Shanghai, China
Magnetic stirring apparatus IKA, Germany
Fluoride ion selective electrode Shanghai Russell, China
Saturated calomel electrode Shanghai Russell, China
Deionized water machine
; Sartorius, Germany
Experimental technique:
1. the FPHA finished product with embodiment 1 is an example, with its compacting in flakes, obtains the FPHA sheet, and (Porcine hydroxyapatite, PHA) sheet is as negative control with biogenetic derivation hydroxyapatite that not fluorine-containing Os Sus domestica spongy bone makes.At each group tabletting surface seeding MG63 cell, use the growing state of scanning electric mirror observing cell, and utilize CCK-8 and ALP test kit to measure cell quantity and the ALP expression of inoculation back in the time of the 3rd, 7,10 day behind the γShe Xianmiejun on the tabletting surface.The result is like Fig. 7, shown in 8 and 9.
The scanning electron microscope of Fig. 7 shows; The MG63 cell is as the active cell of a kind of judge biomaterial commonly used, after FPHA tabletting surface is cultivated three days, and MG63 cell queueing discipline; Form is fusiformis or similar round; The size basically identical meets the form of normal person's osteosarcoma MG63 cell line, be inoculated in the FPHA surface after upgrowth situation good.
Mensuration result to 3,7,10 days each time point ability of cell proliferation sees Fig. 8; The OD value that FPHA and PHA respectively organize cell had tangible growth from 3 days to 7 days; Change not obvious and compared the OD value with 7 days in 10 days; Prompting MG63 cell strain propagation situation on the FPHA tabletting meets the growth curve of MG63 cell strain, is exponential phase of growth at 3 ~ 7 days promptly, gets into plateau after 7 days.In each time point, the average OD value of FPHA group cell climbing speed is high slightly than the PHA group, explains that adherent cell enlargement speed is higher than PHA on the FPHA tabletting, and FPHA promotes cell increment effect to be better than PHA, and grows with time, and difference is more remarkable.
Mensuration result to 3,7,10 days each time point emiocytosis ALP sees Fig. 9; The result shows: the ALP value that FPHA and PHA respectively organize emiocytosis all has rising from growing with time, and FPHA group cytoactive all is higher than the PHA group in each time point, and along with prolong observing time; The PHA group is different increasing with FPHA group OD value difference; Prompting MG63 cell strain cytoactive on the FPHA tabletting is higher than PHA, and along with the growth of time, cytoactive difference is more remarkable.
In sum, FPHA is more having superiority than PHA aspect human body cell adhesion, propagation, differentiation and the expression.
2. measure the fluor releasing quantity that in DMEM low sugar acid medium, soaks dissolving back FPHA and PHA tabletting respectively with the fluorion activity meter.
Table 4: the fluor releasing quantity (unit: μ g) of tabletting in acid medium
|
3 days |
7 days |
10 days |
FPHA |
43.1±0.9 |
32.1±1.6 |
28.1±2.3 |
PHA |
20.0±1.2 |
22.2±1.7 |
29.1±0.8 |
Shown in the table 4, along with the increase of fluorinion concentration, the negative logarithm of fluorinion concentration reduces gradually, and the absolute value of equilibrium potential also reduces thereupon.FPHA and PHA tabletting record the equilibrium potential of each solution and see table according to the corresponding fluorinion concentration that regression equation Y=0.1068X-2.8344 obtains after the dissolving fully in acid medium; It is thus clear that the burst size of the fluorine that records as the PHA of negative control is trace very, almost can ignore.
Table 5: the equilibrium potential that tabletting records after in the neutral culture medium of DMEM low sugar, soaking 3,7,10 days (mean ± standard deviation, mV)
|
Equilibrium potential (mean ± standard deviation, mV) |
-lgF-(ppm) |
F-(ppm) |
FPHA |
16.0±1.4 |
-1.1 |
13.5 |
PHA |
65.3±6.3 |
4.1 |
7.31×10
-5 |
Table 5 shows, the equilibrium potential absolute value of FPHA group is all greater than the equilibrium potential of PHA or gap is little with it, and prompting FPHA does not have tangible fluorine to discharge in neutral culture medium.
After the osseous tissue substitution material implants, material generation bone remodeling, this process comprises the degraded of material and the formation of new bone.The degraded of material comprises the decomposition and absorption of dissolving and the cells in vivo of material in body fluid to material, and hydroxyapatite class bone-grafting material dissolubility under neutral environment is lower, so main still through the degraded of osteoclast metabolism.Research shows that osteoclast is similar with the degraded of body internal skeleton mineral for the degraded of external calcium phosphate embedded material: osteoclast closely contacts the back with material and forms the enclosed area; Secrete hydrion in this differentiation and form local acid environment; PH value is lowered to 4~5, thereby causes material degradation.
Visible from experimental result; FPHA releasing fluoride ion not basically in neutral culture medium; And under sour environment, have tangible fluorine to discharge; Can think among the FPHA fluorion can along with osteoclast to its long-term degradation metabolism and slowly constant release come out, it is too fast and influence Expected Results the back rate of release can not occur implanting.
Three, zoopery
Key instrument equipment:
Superpure water machine 631UF, Sartorius, Germany
Vortex oscillation device VORTEX-GENIE2G560E, Scientific Industries, the U.S.
Slow machine Falcon50k, Silfradent, Italy
Annular is got bone drill Ф 8mm, Helmut Zepf, Germany
Micro-CT Inveon MM Gantry-STD CT camera, Siemens, Germany
Three-dimensional reconstruction system Inveon Acquisition Workplace V1.2, Siemens, Germany
The micro-CT pattern analysis Inveon Research Workplace V2.2.0 of system, Siemens, Germany
Computer automatic dehydration machine YABO600, Changzhou Ya Bo Electronics Equipment Co., Ltd, China
The microtome Leica SP1600 of sclerous tissues, Leica, Germany
Fluorescence is just being put microscope Axioskop40, Zeiss, Germany
IMAQ video camera AxioCam HRc, Zeiss, Germany
Image analysis system AxioVs40AC V4.5.0.0, Zeiss, Germany
Method:
Select 26 bull Sprague-Dawley rats, be divided into three groups at random, 6 of blank control group; Each 10 of PHA group and FPHA groups; Prepare the circular complete damaged model of thick single critical bone of diameter 8mm in each rat skull, the PHA group is implanted Os Sus domestica source hydroxyapatite, and (porcine hydroxyapatite is PHA) as contrast; FPHA group implant fluoridize Os Sus domestica source hydroxyapatite (fluorinated porcine hydroxyapatite, FPHA).FPHA in 0.25mol/L NaF solution soaking 24 hours, places Muffle furnace to be heated to 700 ℃ by PHA afterwards, and heating rate is 10 ℃, and keeps heating 3 hours at 700 ℃, and blank control group is not placed material, directly stitching.The experimental session animal does not have situation such as heating, infection, fluorosis to take place.Get half number animal in the 1st week of postoperative, the 4th week, the micro-CT live body scanning of the 8th week row for every group, put to death in the 8th week at last and get BIAO and BEN, utilize micro-CT pattern analysis system that three-dimensional reconstruction image is carried out the credit of bone morphometry and analyse.Second half animal is got the parallel microscopic CT scanning of BIAO and BEN in putting to death in the 4th week of postoperative, and BIAO and BEN is made the section of undecalcified sclerous tissues with reference to three-dimensional reconstruction image and in the descending histological observation of microscope, the result sees Figure 10 together with organizing in the 8th week.
(1) the micro-CT live body scanning three-dimensional reconstructed image of each processed group rat of different time points is shown in figure 10.
During the 1st week, the damaged edge of blank control group is smooth, does not see skeletonization.It is smooth that PHA group and FPHA organize damaged edge, and obvious embedded material image is not seen by damaged central authorities, and the part embedded material is located to develop on the edge of, and density is lower, does not link to each other with host bone.
During the 4th week, the damaged edge of blank control group owes smooth, at damaged edge the minute quantity skeletonization is arranged.PHA group and FPHA organize damaged edge original embedded material low-density shadow picture and disappear, and semilune area of new bone image appears in the bilateral edge, and density is high, closely links to each other with host bone, has the gap between part area of new bone and the host bone; There are 3 in 5 laboratory animals of FPHA group in the visible highdensity dystopy skeletonization image of damaged central authorities, do not link to each other with peripheral area of new bone.
During the 8th week, all processed group are not all seen damaged bony union.The damaged edge of blank control group owes smooth, and the edge skeletonization increases when all than the 4th slightly.PHA and FPHA organize damaged bilateral area of new bone to be increased on the basis of the 4th all skeletonization to some extent, and density does not have significant change, gap smaller between area of new bone and the host bone or disappearance.Area and density that FPHA organizes damaged central dystopy skeletonization increase, and with the semilune area of new bone at damaged edge the trend that is connected are arranged.
And all do not find the dystopy skeletonization in blank control group, PHA group.Therefore, the implantation of fluorion has the dystopy of promotion ossification.The dystopy skeletonization is meant in the middle of the soft tissue of noncontact osseous tissue, osteoblast differentiation occurs and forms osseous tissue.FPHA compares common biological apatite, has stronger dystopy ossification, explains that its induced dry-cell is divided into osteoblast and generates the effect of osseous tissue stronger.
(2) each processed group rat skull BIAO and BEN undecalcified sclerous tissues sections observation of different time points is like Figure 11,12, shown in 13 and 14.Wherein, the 4th when week all processed group defect of skull all do not see bony union.The blank control group defect is seen the loose fiber connective tissue, and there is a small amount of skeletonization at damaged edge.The down visible area of new bone of high power lens is formed by damaged edge, links to each other with host bone, and the area of new bone bone lacuna is bigger.FPHA group and PHA group defect have loose fiber connective tissue parcel embedded material, and the material multidigit is in damaged edge, and damaged central material is less, and area of new bone mainly appears at damaged edge embedded material and concentrates the place.Distinguishablely under the high power lens go out nattier blue osteoid and bolarious area of new bone holds embedded material, include a large amount of bone lacunas.There is area of new bone directly to link to each other between the embedded material, forms the bone trabecula shape structure of apertured.The host bone at area of new bone and damaged edge has the bone bridging to connect.Embedded material is not seen disintegrate.
All processed group defect of skull are not seen bony union yet during the 8th week.The blank control group defect is a large amount of feltwork connective tissues, and damaged edge skeletonization amount increases.The host bone at area of new bone and damaged edge can not be differentiated under the high power lens, and bone lacuna diminishes.FPHA group and PHA group defect have feltwork connective tissue parcel embedded material, and embedded material reduces when all than the 4th, and damaged edge skeletonization is more; Embedded material in the visible down three groups of experimental grouies of high power lens is all by the area of new bone parcel, and osteoid reduces.The interstructural hole of bone trabecula shape diminishes, and more shows fine and close.Have area of new bone directly to be connected to lamellar between material granule, bone lacuna reduces and diminishes, the material granule part disintegrate of area of new bone parcel.It is thus clear that FPHA has the skeletonization effect similar with clinical biogenic HA commonly used.
(3) the Micro CT live body of each processed group rat of different time points scanning area of new bone volume and surface area are shown in Figure 15 and 16.
During the 1st week, blank control group does not almost have area of new bone, and area of new bone volume and surface area data are almost 0, and the PHA group all has obvious area of new bone to generate with the FPHA group, and wherein the FPHA group is organized apparently higher than PHA.
4th, in 8 whens week,, blank control group, PHA group, FPHA group all detect the generation of area of new bone, and wherein area of new bone volume and surface area data are the FPHA group and are higher than blank control group and are higher than PHA and organize.
At variant time point, the FPHA group explains that all being higher than PHA group and blank control group aspect area of new bone volume and the area of new bone surface area FPHA osteogenic ability is higher than PHA.The comparison of relative the 4th, 8 all skeletonization amounts, FPHA has clear superiority when first week, and is visible, and it is more early faster that FPHA induces area of new bone to generate after implantation.