CN102805879B - Preparation method for biogenic fluorinated porcine hydroxyapatite (FPHA) bone substitution material - Google Patents
Preparation method for biogenic fluorinated porcine hydroxyapatite (FPHA) bone substitution material Download PDFInfo
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- CN102805879B CN102805879B CN201210254493.2A CN201210254493A CN102805879B CN 102805879 B CN102805879 B CN 102805879B CN 201210254493 A CN201210254493 A CN 201210254493A CN 102805879 B CN102805879 B CN 102805879B
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Abstract
The invention discloses a preparation method for a biogenic fluorinated porcine hydroxyapatite (FPHA) bone substitution material. The method comprises the following steps of: 1) sawing cancellous bone of the lower section of thighbone of swine adults into bone blocks, and drying for later use, wherein the cancellous bone of the lower section of the thighbone of the swine adults is used as a raw material; 2) heating the bone blocks and sintering in the air, cooling at room temperature, cleaning completely and drying to obtain porcine hydroxyapatite; 3) soaking the porcine hydroxyapatite in a fluorine-containing solution for 12 to 24 hours, taking out and drying; and 4) placing the porcine hydroxyapatite which is treated in the step 3) in aerobic environment, heating to 700 to 800 DEG C, heating for 2 to 3 hours, cooling, washing, drying, and grinding to obtain powder, namely the biogenic FPHA bone substitution material. According to the method, the thighbone of swine is used as the synthesis raw material, and the bone substitution material which is obtained after physical treatment and chemical fluoridation has a grid structure which is suitable for cell growth, contains an appropriate amount of fluorine, can meet the repairing requirement of clinical bone defect, or can be used as a cell scaffold to be applied to bone tissue engineering, and is wide in source, and low in price.
Description
Technical field
The present invention relates to a kind of preparation method of fluoridizing hydroxyapatite bone alternate material, relate in particular to the preparation method that a kind of biogenic is fluoridized hydroxyapatite bone alternate material.
Background technology
Hydroxyapatite (Hydroxyapatite, hereinafter referred HA) be the most basic inorganic constituents of organism osseous tissue, there are a lot of good characteristics, the similarity of HA and bone mineral composition for example, can promote the Osteoblast Differentiation of stem cell, form nervus vasculairs that strong bone-CaP biomaterial combination interface and guiding the close on nature bone characteristic such as grow into.In medical domain, the materials such as phosphate, calcium salt of take carry out people to be applied as the synthetic pure HA of chemical process once has more in the past, as the filling of a small amount of bone defect repair, bone cyst bone cavity, implant surface coating etc.Yet pure HA comes with some shortcomings for sclerous tissues displacement, undesirable such as physical and mechanical properties, fragility is large etc., especially compare with the biogenic HA occurring afterwards, bone formation performance a little less than, thereby greatly limited its application, substantially by biogenic HA, replaced clinically at present.
Different from synthetic HA is, biogenetic derivation HA be take biological bone as synthesis material, by physics or chemical method removal, wherein can cause the bone alternate material obtaining after the Organic substance of human immunity rejection, its main component is HA, 3-D solid structure is very similar to nature bone, and contains the minor inorganics such as distinctive Ca, Mg, Zn, Cl in nature bone.After the HA bone alternate material of the damaged place of bone implantable bioartificial source, its basis HA can be used as raw material and the space supporting structure in new bone formation process, measurements of the chest, waist and hips porous network structure is more conducive to that human body is osteoblastic to be adhered to and close on osseous tissue blood vessel, neural growing into, and its constituent very similar to nature bone is more conducive to the formation of new bone and the reconstruction of bone-grafting material.Therefore biogenetic derivation HA compares and has absolute advantages with synthetic HA in bone formation rate, skeletonization amount and area of new bone quality, has become current the most conventional bone-grafting material clinically.But the preparation quality of biogenetic derivation HA is often difficult to control, the temperature of preparation is too low, and the organic principle in biological bone is not divisible; The temperature of preparation is too high, there will be carbanion disappearance, and crystalline phase changes, and HA decomposes other materials of production.
The molecular formula of HA is Ca
10(PO
4)
6(OH)
2, Ca wherein
2+, PO
3-, OH
-position all can be substituted by different kinds of ions, so scholar starts to explore some inorganic elementss that adulterate in HA, thereby improves physical and mechanical properties and the whole biological activity of material.Although studies confirm that, take in the excessive meeting of fluorine and cause toxic reaction.But, fluorine is the trace element of needed by human, in the growth and development process of bone, tooth, play an important role, can promote differentiation and the breeding of osteocyte, promote formation and the growth of HA crystal, the mineralising of Bing Dui sclerous tissues plays a driving role, and can improve sclerotin treatment osteoporosis, so it receives much concern thereby there is bibliographical information to increase to a certain extent fluorion intake at aspects such as prevention of osteoporosis diseases.In cell culture experiments, find propagation and alkaline phosphatase activities that fluorion can stimulating osteoblast.Fluoridize the same raising of finding osteoblast bone formation performance in the rat that water raises.Meanwhile, fluorine also has the effect of antibacterial bacteriostatic.The fluorination of drinking water Yi Bei U.S. classifies disease prevention and control center one of 20th century ten big bus health achievement as at present, and obtaining affirming of World Health Organization (WHO) and some other whole world and provincialism health and dentistry tissue, Guangdong Province government has also proposed the project plan about tap water fluoridize.
Existing studies confirm that there is group disappearance, the defects such as Heterogeneous Permutation in the OH group in biogenetic derivation HA in crystal.Wherein, the position of disappearance can be captured by fluorine, the OH of Heterogeneous Permutation is more easily substituted by fluorine, the existence of these defects has increased the alternative probability of fluorine, F substitutes the OH group in HA, can make the dissolubility of apatite reduce, mechanical performance is improved, the ability simultaneously with releasing fluoride ion, make it have stronger bone guided, inducibility and anti-microbial property, obtain good biology performance, more meaningful in only containing the body fluid of low dose fluoride especially, be also one of advantage of the synthetic FPHA of biogenetic derivation HA.It is abundant that biogenetic derivation HA synthetic method has source, cheap, effectively simple, avoid immunogenicity, and FPHA has retained the biological framework of HA, retaining some the favourable trace element in animal body, synthetic FPHA will more be conducive to growing into of osteocyte, having stronger osteoinductive, is a developing direction of following preparation FPHA.
Yet, the technology of preparing at present biogenetic derivation HA mainly rests in foreign corporation's hands, and only have pure biogenic HA to drop into clinical use, China does not still have domestic bone grafting bone alternate material, the biogenetic derivation HA that adopt import clinically in native land more, the Bio-oss that for example Switzerland Co., Ltd produces, biological bone raw material used is Os Bovis seu Bubali.But the price of imported product is very expensive, the purchase price of the Bio-oss that for example Switzerland Co., Ltd produces is 2000 yuan every gram, and domestic patient will defray enormous cost and could use this product to treat.Thereby China needs a kind of biogenetic derivation HA that can substitute Bio-oss badly at present, to reduce patient's treatment cost.
In addition, although existing research has proved the principle that OH group in the biogenetic derivation HA disappearance position in crystal can be substituted by fluorine, fluorine-containing bone alternate material is compared in many aspects in theory common biogenic HA and is all had advantage, but actually still there are a lot of indefinite technology blind spots while preparing fluorion implantable bioartificial source HA, as indefinite in material processed details, detailed preparation process is as sintering bone piece position, size, form, temperature, heating rates etc. are difficult to learn optimum data, academia there is no clear and definite conclusion to fluorine implant concentration etc., therefore, there is not yet the technology report of fluoridizing biogenic HA bone alternate material.
Summary of the invention
The object of this invention is to provide a kind of biogenic and fluoridize the preparation method of hydroxyapatite bone alternate material, the method is usingd pig femur as synthetic raw material, the network both in the bone alternate material that pig femur is obtained after physical treatment and chemical fluoridize with applicable Growth of Cells, and contain appropriate fluorine, can meet the damaged reparation of bone clinically, or be applied to bone tissue engineer as cytoskeleton, there are again the national conditions of meeting, wide material sources, lower-price characteristic.
Object of the present invention is achieved through the following technical solutions: a kind of biogenic is fluoridized the preparation method of hydroxyapatite bone alternate material, comprises the following steps:
(1) draw materials: take the femoral inferior segment spongy bone of pig is raw material, is sawn into 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, then room temperature cleans up after cooling, dry, obtains Os Sus domestica hydroxyapatite (PHA);
(3) soak: Os Sus domestica hydroxyapatite soaks 12 ~ 24 hours in fluorine-containing solution, take out, dry;
(4) reacting by heating: the Os Sus domestica hydroxyapatite after step (3) is processed is placed under aerobic environment and is warming up to 700 ~ 800 ℃, and in this temperature range, heat 2 ~ 3 hours, cooling afterwash, dry, pulverize, obtains biogenic and fluoridizes hydroxyapatite (FPHA) bone alternate material.
In common animals, the similarity the highest (referring to table 1) of Os Sus domestica and people's bone.And the inventor studies discovery, the femoral inferior segment spongy bone of pig has the micropore that is suitable for Growth of Cells, and meanwhile, its macroscopical mesh-like structure is also conducive to flowing and perfusion of blood, and in preparation, spongy bone is beneficial to shaping, clinically also easy operating.Thereby can adopt the femoral inferior segment spongy bone of pig 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 | ++ | ++ | +++ | + |
Note :+low similar, ++ moderate is similar, +++ highly similar
But the organic matter containing due to biological bone can cause the rejection in human body, while therefore using clinically, must take measures to remove completely.Find after deliberation, spongy bone is when 700 ℃ of left and right sintering, and the organic matter in bone almost can be removed completely, and higher than the high temperature sintering of 1000 ℃, just along with the difference of sintering time, there is decomposition in various degree in HA, produces tricalcium phosphate (β-TCP), and its bone conductibility is weaker than HA, and absorption rate is fast compared with HA, can affect bone grafting effect, therefore, the sintering temperature of Os Sus domestica is controlled to 700 ~ 900 ℃.And Os Sus domestica HA 700 ~ 900 ℃ of left and right, mix in the FPHA that fluorine sintering obtains after 1 ~ 2 hour almost without organic residual, without β-TCP and other unformed apatite, if but continue heating, or excess Temperature, can cause the carbonate in carbonated hydroxyapatite to decompose, too low or the too short fluorion of sintering time of temperature is difficult to chemical mode is combined with PHA, can not reach the alternative object of fluorine.Therefore, in order to preserve this carbonate as far as possible, make it to preserve the advantage of biogenic HA, in the present invention, select the temperature of 700 ~ 800 ℃ as the treatment temperature of step (4) reacting by heating.Both preserve carbonate, guaranteed again the doping of fluorine.
The volume size of the bone piece described in step of the present invention (1) is 1 * 1 * 0.5cm
3, be beneficial in subsequent step, bone piece is heated evenly, so that Organic substance is removed completely, fluorion is evenly implanted.
In step of the present invention (1), bone piece is at room temperature dry, can not do other special handlings.As for improving the dry efficiency of bone piece, can adopt the mode of strengthening circulation of air to be dried.
In step of the present invention (3), the used in amounts of fluorine-containing solution meets the absorption requirement of PHA, normally adopts excessive Fluorinse to soak, and for example every 100ml Fluorinse soaks 1g PHA.Bone piece itself has loose structure, and Fluorinse did not have bone piece can reach even contact immersion.
Fluorine-containing solution in step of the present 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.
In intensification sintering in step of the present invention (2), temperature is to rise to 700 ~ 900 ℃ from room temperature, the heating rate of temperature is generally advisable to be no more than 10 ℃/min, because heating rate is too fast, easily cause Organic substance moment burning, local temperature is too high, causes obtained intermedium crystalline phase to change.
Similarly, the heating rate of the heating-up temperature in step of the present invention (3) is generally advisable to be no more than 10 ℃/min, avoids that heating rate is too fast causes the burning of Organic substance moment to cause local temperature too high, prevents that obtained intermedium crystalline phase from changing.
What in step of the present invention (3) and (4), relate to dryly carries out below at 80 ℃.
In step of the present invention (1) ~ (4), after dry, all need semifinished or finished goods to preserve every wet, avoid product to make moist, affect semi-finished product in the result of use for the treatment of effect and the finished product of each step.
The present invention compared with prior art has following beneficial effect:
(1) the present invention be take Os Sus domestica as main synthetic raw material, and its macrostructure, microstructure aspect are the most similar to people's bone, and the similarity of composition, bone density and people's bone is only second to Os Canitis.After substituting, F obtains FPHA.Experiment confirms, this FPHA has retained the biological framework of HA, and retaining some the favourable trace element in animal body, and compare with pure biogenic HA, will more be conducive to growing into of osteocyte, there is stronger osteoinductive, new osteogenesis better effects if, range of application is more extensive, can better be applied to the reparation that osseous tissue is damaged, the support of bone tissue engineer builds, implant surface coating etc.
(2) the present invention be take Os Sus domestica as raw material, more extensive than Os Bovis seu Bubali in its source of China, cheap, be conducive to reduce the cost of raw material, and conflict and medical-risk that bovine spongiform encephalopathy Os Bovis seu Bubali etc. brings without taking precautions against with Hinduism etc. is religious, make application wider of product, simultaneously, be conducive to simplify preparation technology, thereby can greatly reduce the cost of FPHA.
For example, the Bio-oss that the Switzerland Co., Ltd of the import of using at present produces, its purchase price is 2000 yuan every gram, and by the Os Sus domestica of existing market valency purchase, according to the inventive method, be prepared, the Os Sus domestica of every 150 yuan can make 15g biogenic FPHA, 100 yuan of its every gram cost less thaies, and selling price also can be far below the selling price of Bio-oss.Visible, use of the present invention can fundamentally reduce patient's treatment cost, have sizable market competitiveness.
(3) the present invention is by the concentration of adjustment fluoride aqueous solution to make the alternative FPHA of variable concentrations F, and technique is very simple, is easy to realize the production of scale.
(4) the present invention also can realize discarded or only limited to the cycling and reutilization of edible Os Sus domestica in the past, turns waste into wealth.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is further described, described structure and the performance of fluoridizing biogenic hydroxyapatite bone alternate material that the present invention may be better understood.
Fig. 1 is the FPHA configuration of surface figure observing under the scanning electron microscope (SEM) of FPHA, and wherein A is SEM500 times, and B is SEM2000 times.
Fig. 2 is the PHA configuration of surface figure observing under the scanning electron microscope (SEM) of PHA, and wherein A is SEM500 times, and B is SEM2000 times.
Fig. 3 is the PHA configuration of surface figure observing under the scanning electron microscope (SEM) of contrast PHA, and wherein A is SEM500 times, and B is SEM2000 times.
Fig. 4 a is X-ray diffraction (XRD) figure, and wherein A is FPHA group, and A heated is for soaking PHA group, and B is PHA, and B heated is contrast PHA group, the HA that C is synthetic.
Fig. 4 b is X-ray diffraction (XRD) figure, and wherein A is FPHA group, and A heated is for soaking PHA group, and B is PHA, and B heated is contrast PHA group, the HA that C is synthetic.
Fig. 5 a is Fourier's infrared spectrum (FTIR) figure, and wherein, A is FPHA group, and A heated is for soaking PHA group, and B is PHA, and B heated is contrast PHA group, the HA that C is synthetic.
Fig. 5 b is Fourier's infrared spectrum (FTIR) figure, and wherein, A is FPHA group, and A heated is for soaking PHA group, and B is PHA, and B heated is contrast PHA group, the HA that C is synthetic.
Fig. 6 is the dissolubility that solid titration method records PHA and HA.
Fig. 7 is that MG63 cell is cultivated the growing state scanning electron microscope (SEM) photograph (SEM) after three days on FPHA sheet surface, and wherein A is the Electronic Speculum figure of 200 times of mirrors, and B is the Electronic Speculum figure of 1000 times of mirrors, and C is the Electronic Speculum figure of 2000 times of mirrors.
Fig. 8 is that MG63 cell is at the different time propagation situation comparison diagram on FPHA and PHA tabletting surface.
Fig. 9 is that MG63 cell is at the different time cell viability comparison diagram on FPHA and PHA tabletting surface.
Figure 10 is the micro-CT vivo scan three-dimensional reconstruction image of each processed group rat, wherein, and when A is the 1st week, when B is 4th week, when C is the 8th week.
Figure 11 is the optical microscope figure under 50 times of mirrors of blank group rat skull exemplar undecalcified sclerous tissues's section, and wherein, the section of top is sclerous tissues's section in the 4th week, and the section of below is sclerous tissues's section in the 8th week.
Figure 12 is the optical microscope figure under 50 times of mirrors of PHA group rat skull exemplar undecalcified sclerous tissues's section, and wherein, the section of top is sclerous tissues's section in the 4th week, and the section of below is sclerous tissues's section in the 8th week.
Figure 13 is the optical microscope figure under 50 times of mirrors of FPHA group rat skull exemplar undecalcified sclerous tissues's section, and wherein, the section of top is sclerous tissues's section in the 4th week, and the section of below is sclerous tissues's section in the 8th week.
Figure 14 is the section of each processed group rat skull exemplar undecalcified sclerous tissues at the optical microscope figure of 200 times.
Figure 15 is each processed group rat Micro CT vivo scan area of new bone volume comparison diagram.
Figure 16 is each processed group rat Micro CT vivo scan area of new bone surface area comparison diagram.
The specific embodiment
Set forth in further detail content of the present invention with experimental example by the following examples, but following embodiment is just for setting forth content of the present invention, rather than restriction, therefore in the implication suitable with claims of the present invention and any change in scope, all should think to be included in the scope of claims.
Embodiment 1
(1) draw materials: get into pig femoral inferior segment spongy bone, remove most soft tissue, clean, be sawn into approximately 1 * 1 * 0.5cm of size
3bone piece, drying at room temperature is standby.
(2) sintering: the bone piece sintering that heats up in air in Muffle furnace, variations in temperature in programme-control stove, heating rate with 10 ℃/min rises to 800 ℃ from room temperature, then at 800 ℃, maintains sintering 2 hours, takes out after being cooled to room temperature, washed with de-ionized water once, remove the residue on bone piece surface, filter, 80 ℃ dry, obtain Os Sus domestica hydroxyapatite (PHA), then every wet, preserve.
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.25mol/LNaF solution, allow PHA be soaked in excessive NaF solution, after immersion 24h, take out PHA; All PHA in 80 ℃ dry, every wet, preserve.
(4) reacting by heating: the PHA processing through step (3) is placed in to clean Muffle furnace, under aerobic environment, carry out reacting by heating processing, heating-up temperature is warming up to 700 ℃ with 10 ℃/min of heating rate from room temperature, then PHA maintains heating 3 hours in 700 ℃ of temperature, heat treated finishes rear taking-up and repeatedly cleans 1min with deionized water, 80 ℃ are milled into powdery after dry, obtain biogenic and fluoridize hydroxyapatite (FPHA) bone alternate material, every wet, preserve.
Sample is carried out to physicochemical property detection and zoopery, and result is referring to experimental example.
Embodiment 2
Different from embodiment mono-:
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.50mol/LNaF solution, allow PHA be soaked in excessive NaF solution, after immersion 12h, take out PHA; All PHA in 80 ℃ dry, every wet, preserve.
(4) reacting by heating: the PHA processing through step (3) is placed in to clean Muffle furnace carries out reacting by heating processing under aerobic environment, heating-up temperature is warming up to 750 ℃ with 10 ℃/min of heating rate from room temperature, then PHA maintains heating 2.5 hours in 750 ℃ of temperature, heat treated finishes rear taking-up and repeatedly cleans 1min with deionized water, 80 ℃ are milled into powdery after dry, obtain biogenic and fluoridize hydroxyapatite (FPHA) bone alternate material, every wet, preserve.
Sample is carried out to physicochemical property detection and zoopery, and result is referring to experimental example.
Embodiment 3
Different from embodiment mono-:
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.75mol/LNaF solution, allow PHA be soaked in excessive NaF solution, after immersion 20h, take out PHA; All PHA in 80 ℃ dry, every wet, preserve.
(4) reacting by heating: the PHA processing through step (3) is placed in to clean Muffle furnace carries out reacting by heating processing under aerobic environment, heating-up temperature is warming up to 800 ℃ with 10 ℃/min of heating rate from room temperature, then PHA maintains heating 2 hours in 800 ℃ of temperature, heat treated finishes rear taking-up and repeatedly cleans 1min with deionized water, 80 ℃ are milled into powdery after dry, obtain biogenic and fluoridize hydroxyapatite (FPHA) bone alternate material, every wet, preserve.
Sample is carried out to physicochemical property detection and zoopery, and result is referring to experimental example.
Embodiment 4
Different from embodiment mono-:
(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 respectively 18h; All PHA in 80 ℃ dry, every wet, preserve.
Sample is carried out to physicochemical property detection and zoopery, and result is referring to experimental example.
Embodiment 5
(1) draw materials: get into pig femoral inferior segment spongy bone, remove most soft tissue, clean, be sawn into approximately 1 * 1 * 0.5cm of size
3bone piece, drying at room temperature is standby.
(2) sintering: the bone piece sintering that heats up in air in Muffle furnace, variations in temperature in programme-control stove, with the heating rate of 10 ℃/min, from room temperature, rise 700 ℃, then at 700 ℃, maintain sintering 2.5 hours, take out after being cooled to room temperature, washed with de-ionized water once, remove the residue on bone piece surface, filter, 80 ℃ dry, obtain Os Sus domestica hydroxyapatite (PHA), then every wet, preserve.
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml 0.25mol/LNaF solution, allow PHA be soaked in excessive NaF solution, after immersion 24h, take out PHA; All PHA in 80 ℃ dry, every wet, preserve.
(4) reacting by heating: the PHA processing through step (3) is placed in to clean Muffle furnace carries out reacting by heating processing under aerobic environment, heating-up temperature is warming up to 800 ℃ with 10 ℃/min of heating rate from room temperature, then PHA maintains heating 2 hours in 800 ℃ of temperature, heat treated finishes rear taking-up and repeatedly cleans 1min with deionized water, 80 ℃ are milled into powdery after dry, obtain biogenic and fluoridize hydroxyapatite (FPHA) bone alternate material, every wet, preserve.
Embodiment 6
(1) draw materials: get into pig femoral inferior segment spongy bone, remove most soft tissue, clean, be sawn into approximately 1 * 1 * 0.5cm of size
3bone piece, drying at room temperature is standby.
(2) sintering: the bone piece sintering that heats up in air in Muffle furnace, variations in temperature in programme-control stove, with the heating rate of 10 ℃/min, from room temperature, rise 900 ℃, then at 900 ℃, maintain sintering 1 hour, take out after being cooled to room temperature, washed with de-ionized water once, remove the residue on bone piece surface, filter, 80 ℃ dry, obtain Os Sus domestica hydroxyapatite (PHA), then every wet, preserve.
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.05mol/LNaF solution, allow PHA be soaked in excessive NaF solution, after immersion 20h, take out PHA; All PHA in 80 ℃ dry, every wet, preserve.
(4) reacting by heating: the PHA processing through step (3) is placed in to clean Muffle furnace carries out reacting by heating processing under aerobic environment, heating-up temperature is warming up to 700 ℃ with 10 ℃/min of heating rate from room temperature, then PHA maintains heating 3 hours in 700 ℃ of temperature, heat treated finishes rear taking-up and repeatedly cleans 1min with deionized water, 80 ℃ are milled into powdery after dry, obtain biogenic and fluoridize hydroxyapatite (FPHA) bone alternate material, every wet, preserve.
Embodiment 7
(1) draw materials: get into pig femoral inferior segment spongy bone, remove most soft tissue, clean, be sawn into approximately 1 * 1 * 0.5cm of size
3bone piece, drying at room temperature is standby.
(2) sintering: the bone piece sintering that heats up in air in Muffle furnace, variations in temperature in programme-control stove, with the heating rate of 10 ℃/min, from room temperature, rise 750 ℃, then at 750 ℃, maintain sintering 2.5 hours, take out after being cooled to room temperature, washed with de-ionized water once, remove the residue on bone piece surface, filter, 80 ℃ dry, obtain Os Sus domestica hydroxyapatite (PHA), then every wet, preserve.
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.04mol/LNaF solution, allow PHA be soaked in excessive NaF solution, after immersion 24h, take out PHA; All PHA in 80 ℃ dry, every wet, preserve.
(4) reacting by heating: the PHA processing through step (3) is placed in to clean Muffle furnace carries out reacting by heating processing under aerobic environment, heating-up temperature is warming up to 750 ℃ with 10 ℃/min of heating rate from room temperature, then PHA maintains heating 3 hours in 750 ℃ of temperature, heat treated finishes rear taking-up and repeatedly cleans 1min with deionized water, 80 ℃ dry rear with being milled into powdery, obtain biogenic and fluoridize hydroxyapatite (FPHA) bone alternate material, every wet, preserve.
Experimental example:
What take embodiment 1 ~ 4 makes sample as example, respectively it is carried out that physicochemical property detection, cell in vitro are learned and detected, animal vivo test detects.Experiment component is PHA group, contrast PHA group, soaks PHA group and FPHA group, and the Os Sus domestica source property hydroxyapatite (PHA) that wherein PHA group is prepared by high temperature sintering for the present invention, confirms consistent with clinical conventional biogenic HA composition through experiment detection; Contrasting PHA group is to carry out by preparing identical method with FPHA the PHA that reacting by heating is processed gained after deionized water immersion PHA, and PHA organizes and contrasts PHA group all as testing reference with comparative evaluation FPHA physics and chemistry and biology performance; Soak PHA group for PHA soaks but meso sample that sintering is not made in Fluorinse, wherein contain certain density fluorion, but non-chemically implant for physical absorption, be soaked in liquid and can separate out fast loss.
One, physicochemical property
PHA and the FPHA of embodiment 1 ~ 4 of take is example, adopts SEM, XPS, and FTIR, XRD, EDS, TEM, trace solid titrimetry is carried out 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 physicochemical property testing result is as follows:
1. morphology analysis:
The sample of embodiment 2 of take is example, scanning electron microscope as shown in Figure 1,2 and 3 (SEM) testing result, and pattern is similar substantially to FPHA for PHA, is loose porous space network; Under high power lens, can find that the crystal grain of FPHA is for long bar-shaped, form is more regular compared with 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 are all mainly comprised of calcium, phosphorus and oxygen element, also contain a certain amount of carbon and Trace Sodium and magnesium.Its difference is, through fluoridizing the FPHA of gained, contains a certain amount of fluorine.Composition is as shown in table 2 in detail.
Table 2:EDS half-quantitative detection PHA and FPHA composition
3. crystal 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.Due to fluorine implantation, PHA causes its Lattice Contraction, and 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:
If Fig. 5 a and 5b are according to Fourier's infrared spectrum (FTIR) testing result, in PHA and FPHA, all contain carbonate group, this two be carbonated hydroxyapatite.In addition, because fluorion is implanted in PHA lattice, substituted part of hydroxyl, the absworption peak of hydroxyl in FPHA is disappeared.
5. fluorine chemistry state analysis:
The combination that shows fluorine in x-ray electron spectroscopy analysis (XPS) result can reduce, and shows that variation has occurred its chemical state, and its residing chemical micro-environment changes, by Na-F, changes in HA the state with calcium, phosphorus, the multiple interaction between atoms of oxygen into.Concrete delta data is as shown in table 3.Wherein, C represents the HA of synthetic, 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, 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
At present the most situations of bone-grafting material are to be all applied in the region that needs long term maintenance bone grafting space clinically, nonabsorable or to absorb bone-grafting material be slowly the first selection, and while carrying out bone grafting in the aesthstic district of the upper jaw, recommendation absorbs bone-grafting material slowly.Concerning bone-grafting material, the bone alternate material of low solubility has greater advantages.Adopt solid titration method to measure the dissolubility of PHA and HA, as shown in Figure 6, PHA dissolubility, lower than HA, is about 1/4th of HA, illustrates that PHA absorption rate can be lower than HA.And having document to confirm, phosphorus ash stone crystal dissolubility after OH group is replaced by F can further reduce, so FPHA also has suitable advantage aspect dissolubility.
From above-mentioned physicochemical property experimental result, the PHA sample of embodiment 2 is consistent with clinical conventional biogenic HA composition, and main component is HA, contains the inorganic constituents that a small amount of natural bone tissue has simultaneously; FPHA finished product only replaces the OH group gained in PHA for fluorion chemical replacement, 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, 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 test, 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 Fu Yuanming instrument and equipment company limited, China
Gamma radiator Beijing, GM-2 type cobalt-60 gamma new and high technology company limited, China
Electronic balance ME235S, Sartorius, Germany
Superclean bench Kendrol, Germany
CO2 constant-temperature incubation case Shel Lab, the U.S.
Axiovert40 inverted microscope Zeiss, Japan
Electric heating constant-temperature blowing drying box 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
Deionizing water machine
, Sartorius, Germany
Experimental technique:
1. the FPHA finished product of embodiment 1 of take is example, is suppressed in flakes, obtains FPHA sheet, and biogenetic derivation hydroxyapatite (Porcine hydroxyapatite, the PHA) sheet that the not fluorine-containing Os Sus domestica spongy bone of usining makes is as negative control.After γ ray sterilization, at each group tabletting surface seeding MG63 cell, use scanning electric mirror observing cell at the growing state on tabletting surface, and utilize CCK-8 and ALP test kit to measure cell quantity and the ALP expression of inoculation in the time of latter the 3rd, 7,10 days.Result is as shown in Fig. 7,8 and 9.
The scanning electron microscope of Fig. 7 shows, MG63 cell is as a kind of cell of conventional judge biomaterial activity, on FPHA tabletting surface, cultivate after three days, MG63 cell arrangement rule, form is fusiformis or similar round, size is basically identical, meets the form of normal person's osteosarcoma MG63 cell line, be inoculated in FPHA surface after upgrowth situation good.
Measurement result to 3,7,10 days each time point ability of cell proliferation is shown in Fig. 8, the OD value that FPHA and PHA respectively organize cell had obvious growth from 3 days to 7 days, and within 10 days, compared OD value with 7 days, change not obvious, prompting MG63 cell strain propagation situation on FPHA tabletting meets the growth curve of MG63 cell strain, at 3 ~ 7 days, be exponential phase of growth, after 7 days, enter plateau.In each time point, the average OD value of FPHA group cell climbing speed is slightly high compared with PHA group, illustrates that the cell enlargement speed adhering on FPHA tabletting is higher compared with PHA, and FPHA promotes cell increment effect to be better than PHA, and along with the time increases, difference is more remarkable.
Measurement result to 3,7,10 days each time point emiocytosis ALP is shown in Fig. 9, result shows: FPHA and PHA respectively organize the ALP value of emiocytosis from increasing and all have rising along with the time, in each time point, FPHA group cytoactive is all higher than PHA group, and along with extend observing time, PHA group is different increasing with FPHA group OD value difference, cytoactive is higher than PHA on FPHA tabletting for prompting MG63 cell strain, and along with the growth of time, cytoactive difference is more remarkable.
In sum, FPHA is more having superiority compared with PHA aspect human body cell adhesion, propagation, differentiation and expression.
2. with fluorion activity meter, measure and in DMEM low sugar acid medium, soak respectively the fluor releasing quantity that dissolves rear FPHA and PHA tabletting.
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 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 after dissolving completely, record the equilibrium potential of each solution in acid medium and the corresponding fluorinion concentration obtained according to regression equation Y=0.1068X-2.8344 in Table, the burst size of the visible fluorine recording as the PHA of negative control is very micro-, almost negligible.
Table 5: the equilibrium potential that tabletting records soak 3,7,10 days in the neutral culture medium of DMEM low sugar after (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 demonstration, 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 obvious fluorine to discharge in neutral culture medium.
After 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 that dissolving and the cells in vivo of material in body fluid absorbs the decomposition of material, and hydroxyapatite class bone-grafting material dissolubility under neutral environment is lower, so mainly still degrade by osteoclast metabolism.Research shows that osteoclast is similar to the degraded of body internal skeleton mineral for the degraded of external calcium phosphate embedded material: after osteoclast and material close contact, form enclosed area, in this differentiation, secrete hydrion and form local acid environment, pH value is lowered to 4～5, thereby causes material degradation.
From experimental result, FPHA releasing fluoride ion not substantially in neutral culture medium, and under sour environment, have obvious fluorine to discharge, can think in FPHA fluorion can along with osteoclast to its long-term degradation metabolism and slowly constant release out, there will not be implant after rate of release too fast and affect Expected Results.
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
Micro-CT Graphic System Inveon Research Workplace V2.2.0, Siemens, Germany
Computer automatic dehydrator 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
Image acquisition 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 at random three groups, 6 of blank groups, each 10 of PHA group and FPHA groups, in each rat skull, prepare the circular complete thick single critical bone defect model of diameter 8mm, PHA group is implanted Os Sus domestica source hydroxyapatite (porcine hydroxyapatite, PHA) in contrast, FPHA group is implanted and is fluoridized Os Sus domestica source hydroxyapatite (fluorinated porcine hydroxyapatite, FPHA).FPHA, is placed in afterwards Muffle furnace and is heated to 700 ℃ in 0.25mol/L NaF solution soaking 24 hours by PHA, and heating rate is 10 ℃, and keeps heating 3 hours at 700 ℃, and blank group is not placed material, directly sews up.Experimental session animal does not have the situations such as heating, infection, fluorosis to occur.Get half number animal in postoperative the 1st week, the 4th week, the 8th week micro-CT vivo scan of row for every group, finally in the 8th week, put to death label taking originally, utilize micro-CT Graphic System to carry out bone morphometry Epidemiological Analysis to three-dimensional reconstruction image.Second half animal was put to death this parallel microscopic CT scanning of label taking in postoperative the 4th week, together with the 8th week group specimen, with reference to three-dimensional reconstruction image, make the section of undecalcified sclerous tissues and in the descending histological observation of microscope, the results are shown in Figure 10.
(1) the micro-CT vivo scan three-dimensional reconstruction image of each processed group rat of different time points as shown in figure 10.
In the time of the 1st week, the damaged the smooth of the edge of blank group, has no skeletonization.PHA group and FPHA organize damaged the smooth of the edge, and damaged central authorities have no obvious embedded material image, and part embedded material develops in edge, and density is lower, is not connected with host bone.
In the time of the 4th week, the damaged edge of blank group owes smooth, at damaged edge, has minute quantity skeletonization.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 double-side-edge, and density is high, is closely connected with host bone, between part area of new bone and host bone, has gap; In 5 laboratory animals of FPHA group, there are 3 in the visible highdensity ectopic osteogenesis image of damaged central authorities, are not connected with periphery area of new bone.
In the time of the 8th week, all processed group are showed no damaged bony union.The damaged edge of blank group owes smooth, and edge skeletonization slightly increases during compared with the 4th week.PHA and FPHA organize damaged bilateral area of new bone to be increased to some extent on the basis of the 4th week skeletonization, and density is without significant change, the gap smaller between area of new bone and host bone or disappearance.Area and the density of the damaged central ectopic osteogenesis of FPHA group increase, and have with the semilune area of new bone at damaged edge the trend being connected.
And all do not find ectopic osteogenesis at blank group, PHA group.Therefore, the implantation of fluorion has promotion ectopic.Ectopic osteogenesis refers in the middle of the soft tissue of noncontact osseous tissue, occurs osteoblast differentiation and forms osseous tissue.FPHA compares common biological apatite, has stronger ectopic, illustrates that its induced dry-cell is divided into osteoblast and generates the effect of osseous tissue stronger.
(2) each processed group rat skull exemplar undecalcified sclerous tissues sections observation of different time points is as shown in Figure 11,12,13 and 14.Wherein, all processed group defect of skull are showed no bony union the 4th week time.Loose fiber connective tissue is seen at the damaged place of blank group, and there is a small amount of skeletonization at damaged edge.Under high power lens, visible area of new bone is formed by damaged edge, is connected with host bone, and area of new bone bone lacuna is larger.FPHA group and PHA organize damaged place loose fiber connective tissue parcel embedded material, and 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 place.Distinguishablely under high power lens go out nattier blue osteoid and bolarious area of new bone holds embedded material, include a large amount of bone lacunas.Between embedded material, there is area of new bone to be directly connected, form the bone trabecula shape structure with hole.The host bone at area of new bone and damaged edge has bone bridging to connect.Embedded material has no disintegrate.
In the time of the 8th week, all processed group defect of skull have not yet to see bony union.The damaged place of blank group is a large amount of feltwork connective tissues, and damaged edge skeletonization amount increases.Under high power lens, the host bone at area of new bone and damaged edge can not be differentiated, and bone lacuna diminishes.FPHA group and PHA organize damaged place feltwork connective tissue parcel embedded material, embedded material minimizing during compared with the 4th week, and damaged edge skeletonization is more; Embedded material under high power lens in visible three groups of experimental grouies is all wrapped up by area of new bone, and osteoid reduces.The interstructural hole of bone trabecula shape diminishes, more aobvious densification.Between material granule, have area of new bone to be directly connected to lamellar, bone lacuna reduces and diminishes, the material granule part disintegrate of area of new bone parcel.Visible, FPHA has the skeletonization effect similar to clinical conventional biogenic HA.
(3) the Micro CT vivo scan area of new bone volume of each processed group rat of different time points and surface area are as shown in Figure 15 and 16.
In the time of the 1st week, blank group is almost without area of new bone, and area of new bone volume and surface area data are almost 0, PHA group all has obvious area of new bone to generate with FPHA group, and wherein FPHA group is apparently higher than PHA group.
4th,, 8 weeks time, blank group, PHA group, FPHA group all detect the generation of area of new bone, wherein area of new bone volume and surface area data be FPHA group higher than blank group higher than PHA group.
At variant time point, FPHA group is all organized and blank group higher than PHA aspect area of new bone volume and area of new bone surface area, illustrates that FPHA osteogenic ability is higher than PHA.The comparison of relative the 4th, 8 weeks skeletonization amounts, in the time of first week, FPHA has clear superiority, visible, and it is more early faster that FPHA induces area of new bone to generate after implantation.
Claims (1)
1. biogenic is fluoridized a preparation method for hydroxyapatite bone alternate material, it is characterized in that, comprises the following steps:
(1) take pig femoral inferior segment spongy bone is raw material, removes most soft tissue, cleans, and is sawn into approximately 1 * 1 * 0.5cm of size
3bone piece, drying at room temperature is standby;
(2) sintering: the bone piece sintering that heats up under air atmosphere in Muffle furnace, variations in temperature in programme-control stove, heating rate with 10 ℃/min rises to 800 ℃ from room temperature, then at 800 ℃, maintains sintering 2 hours, takes out after being cooled to room temperature, washed with de-ionized water once, remove the residue on bone piece surface, filter, 80 ℃ dry, obtain Os Sus domestica hydroxyapatite PHA, then every wet, preserve;
(3) soak: in the ratio of every 100ml NaF solution soaking 1gPHA, get 1gPHA and be dipped in 100ml0.25mol/LNaF solution, allow PHA be soaked in excessive NaF solution, after immersion 24h, take out PHA; All PHA in 80 ℃ dry, every wet, preserve;
(4) reacting by heating: the PHA processing through step (3) is placed in to clean Muffle furnace, under aerobic environment, carry out reacting by heating processing, heating-up temperature is warming up to 700 ℃ with 10 ℃/min of heating rate from room temperature, then PHA maintains heating 3 hours in 700 ℃ of temperature, heat treated finishes rear taking-up and repeatedly cleans 1min with deionized water, 80 ℃ are milled into powdery after dry, obtain biogenic and fluoridize hydroxyapatite FPHA bone alternate material, every wet, preserve.
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