WO2018035773A1 - 再生医学材料及其制备方法和应用 - Google Patents

再生医学材料及其制备方法和应用 Download PDF

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WO2018035773A1
WO2018035773A1 PCT/CN2016/096604 CN2016096604W WO2018035773A1 WO 2018035773 A1 WO2018035773 A1 WO 2018035773A1 CN 2016096604 W CN2016096604 W CN 2016096604W WO 2018035773 A1 WO2018035773 A1 WO 2018035773A1
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regenerative medicine
medicine material
inorganic substance
hard tissue
tissue repair
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PCT/CN2016/096604
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English (en)
French (fr)
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胡方
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胡方
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Priority to EP16913796.5A priority Critical patent/EP3505195B1/en
Priority to US16/327,734 priority patent/US10828397B2/en
Priority to KR1020197008434A priority patent/KR102258806B1/ko
Priority to PCT/CN2016/096604 priority patent/WO2018035773A1/zh
Priority to JP2018546606A priority patent/JP2019506985A/ja
Priority to CN201610955130.XA priority patent/CN106362214A/zh
Publication of WO2018035773A1 publication Critical patent/WO2018035773A1/zh

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Definitions

  • the invention relates to the field of regenerative medicine, in particular to a regenerative medicine material and a preparation method and application thereof.
  • bioactive glass and glass ceramics The most striking feature of bioactive glass and glass ceramics is that the surface condition changes dynamically with time after implantation into the human body.
  • the surface forms a bioactive hydroxycarbonated apatite (HCA) layer, which provides a bonding interface for the tissue.
  • HCA bioactive hydroxycarbonated apatite
  • Most bioactive glasses are Class A bioactive materials, both osteogenic and osteoconductive, which have good binding to bone and soft tissue.
  • Bioactive glass (BAG) is considered to be Good biomaterials for use in the field of restoration.
  • the use of such restorative materials is not only extremely extensive, but also has irreplaceable magical effects in professional products in many fields, such as skin care, whitening wrinkles, burns, mouth ulcers, gastrointestinal ulcers, skin ulcers, killing fungi.
  • BAG also has a rapid surface reaction; amorphous two-dimensional structure has low strength and fracture toughness; elastic modulus (30-35 MPa) is low, close to cortical bone; and machinable bioglass has good processing properties.
  • an aspect of the present invention provides a regenerative medicine material that promotes soft and hard tissue repair, the regenerative medicine material having a three-dimensional network structure, and being a composite material composed of inorganic substances and organic substances, wherein The mass ratio of the inorganic substance to the organic substance is 2:1 to 4:1;
  • the inorganic substance contains 12.38% SiO 2 , 3-5% Na 2 O, 15-29% CaO, 10-32.5% P 2 O 5 , 1-5% inositol based on the total mass of the inorganic substance.
  • the organic material comprises 30-60% carboxymethyl chitosan and 30-60% sodium hyaluronate based on the total mass of the organic matter.
  • the mass ratio of the inorganic material to the organic matter is 3:1. Further, the weight ratio of calcium to phosphorus in the inorganic substance is from 1.5 to 1.8, preferably 1.67.
  • the inorganic substance is obtained by a sol-gel method using a soybean hull extract as a phosphorus precursor and sintering at a constant temperature of 300 to 700 °C.
  • a cell growth carrier comprising the above-described regenerative medicine material for promoting soft and hard tissue repair.
  • the cell growth carrier is a medical device, in particular at least a portion of an implant.
  • a method of promoting soft and hard tissue repair is provided, wherein the regenerative medical material described in the present invention is used.
  • a regenerative medical material according to the invention for promoting soft and hard tissue repair in the preparation of a pharmaceutical composition, medical device, oral care product, orthopedic product or cosmetic.
  • the regenerative medicine material of the invention can better control the degradation rate of the material, and the degradation rate of the original bioactive glass can be controlled, and the degradation rate of the new bone implant material can be synchronized in the application field of the artificial bone material, and the bone structure cannot be reached by overcoming the new bone.
  • the regenerative medicine material of the present invention is of great significance when applied to bone repair, spinal and bone defects, oral bone implantation, and the like.
  • the degradation rate of the regenerative medicine material can be improved.
  • the materials prepared by the conventional preparation method have a small range of bioactive components, and the degradation rate is usually limited by reducing the phosphorus content.
  • the material prepared by the conventional method usually uses phosphoric acid, ethyl phosphate or the like, and its compatibility with the precursor of calcium (for example, calcium nitrate) is poor, and it is easy to cause separation of the precipitate. Selecting a more toxic ethylene glycol reduces the concentration of the precursor, which consumes a lot of energy and time in the process of treating the solvent, and it is difficult to achieve standardized and large-scale production.
  • the phosphorus precursor of the invention can be effectively co-dissolved with a calcium precursor such as calcium nitrate, the solvent is water, ethanol or a mixture thereof, the toxicity is small, the solvent removal temperature is low, and the room temperature is realized by changing precursors such as calcium and sodium.
  • the gel is formed under the condition to achieve various physical and chemical indexes of the bioactive glass at 600 °C. Overcome the shortcomings of traditional methods and achieve standardization and large-scale production.
  • 1% to 10% of silicon atoms have a high coordination number of 5 or more, and these highly-coordinated silicon are formed under normal pressure.
  • High coordination silicon has a certain influence on the structure and properties of the material. For example, high coordination silicon will shift the ultraviolet absorption peak.
  • the silicon coordination number is changed from 4 to 6, mainly in the form of Si-OP or Si-O-Si, and as the content of P increases, 6
  • the coordination of silicon increases, after heat treatment, the glass material will crystallize, and the solid materials of silicon with high coordination silicon atoms at medium and low temperature and normal pressure have not yet appeared.
  • the regenerative medicine material of the invention is superior to the physical and chemical indicators and biological indicators of the bioactive glass.
  • the material achieves high-efficiency energy-saving at 600 °C, large-scale production with controllable quality, and changes the disadvantages of high energy consumption and low product qualification rate of the original 1700-1900 °C calcination.
  • other medical materials are added, and it is widely used in the field of biological therapy.
  • the ratio of calcium to phosphorus is relatively consistent with the human skeleton, and is, for example, about 1.67.
  • the three-dimensional network structure and pore size formed can be consistent with the pore size of human bones and soft tissues.
  • the biological activity, stability and degradation rate of the material can be controlled.
  • the material is prepared at a low temperature, It can be loaded with bioactive molecules, such as proteins, antibiotics, chemotherapeutic drugs, and is porous, can be used for drug loading and controlled release, and the phosphorus precursor used is a natural substance, which is relatively less toxic to traditional phosphorus precursors, thus Improve the biocompatibility of materials.
  • the material rapidly forms hydroxyapatite on the surface in simulated human body fluid (SBF).
  • This material can overcome the poor plasticity of traditional bioactive glass. Forming an adjustable amorphous material, an amorphous structure.
  • the other bioactive glass is basically a crystal, and the regenerative medicine material of the invention is amorphous, the particles of the material are uniform, the biocompatibility of the material is stable, the dispersibility is good, and the stability is strong.
  • the compression strength and degradation rate in the artificial bone are all controllable.
  • the regenerative medicine material of the present invention contains Si, Ca, Na, and P ions, and the ratio of each ion is identical to that of human bone tissue. These ions are uniformly distributed in the multi-pore diameter of the high-temperature calcined SiO 2 , which naturally causes an eight-step surface reaction between the material and the body fluid.
  • the first step the Na + and K + ions in the material are evenly distributed in the multi-aperture SiO 2 (the pore size is almost the same as the human bone diameter), they encounter body fluids, and the H + and H 3 O + ions exchange rapidly.
  • the pore size is almost the same as the human bone diameter
  • OH - is a negative charge
  • the adsorption of bone tissue and soft tissue cells, growth factors, collagen, and other substances to the growth of ordered pore forming machine body block structure is grown.
  • the regenerative cells express genes, grow in order, and form bone guiding and bone conduction ability.
  • the second step Si-O-Si bond, interrupted by the solution, forming a lot of Si-OH outside the interface;
  • the third step the polymerization of Si-OH will form a porous limb layer of SiO 2 ; it combines with different kinds of proteins to form high-density protein adsorption through hydrogen bonding and ionic amine bond (-Si-OH 3 N + -).
  • a silica sol layer and a hydroxycarbonated apatite layer are formed, and the gray stone layer has a high surface area and is suitable for adsorbing a large amount of biomolecules, thereby promoting an extracellular response.
  • the new bone adsorbs more biomolecules than the silica sol layer with a lower negative charge.
  • the fourth step high coordination of silicon atoms 6, silicon atoms 4 and human potassium exchange, forming a stable three-dimensional network solid state mechanism, changing the original silicon atoms to a free state. It can be naturally exchanged with human body chemical components to form a carrier for crawling replacement. High-coordination silicon will transfer the ultraviolet absorption peak and play an antioxidant role.
  • Step 5 Ca 2+ and PO 4 3- source materials or sources and solutions, aggregate on the SiO 2 -rich limb layer to form a CaO-P 2 O 5 amorphous phase layer; regulate the nascent cells by the ratio of P Synchronized with the degradation of the original implant.
  • Step 6 As OH- and CO 3 2- are extracted from the solution, the CaO-P 2 O 5 non-oriented phase layer will be converted into carbon-containing hydroxyapatite (HCA) polycrystals, which are adsorbed on wounds, ulcers and soft tissues. On the surface, it promotes cell proliferation, promotes wound healing, and repairs wounds without scars.
  • HCA hydroxyapatite
  • Step 7 Promote the orderly growth of cell proliferation, promote the formation of hair follicles, promote the growth of hair follicles, and achieve the effect of reducing scars.
  • Step 8 Ulcer wounds, especially oral ulcers, cervical erosion are caused by anaerobic bacteria.
  • the regenerative medicine materials of the present invention can form a weak alkaline environment, let anaerobic bacteria dehydrate and die, and simultaneously inhibit the growth and promotion of anaerobic bacteria. Repair and proliferation of new cells.
  • the regenerative medicine material of the present invention not only has various properties suitable for the human body, but also has a more consistent composition and content relationship with the human body, thereby being more suitable for tissue repair.
  • XRF X-ray fluorescence spectrum
  • FIG. 3 is a SEM-EDXS diagram of an exemplary regenerative medical material of the present invention. A- Before deposition, after b-SBF deposition (14d).
  • Regenerative medical material of the present invention is an MTT value of an exemplary regenerative medical material of the present invention and its extract interacting with osteoblasts.
  • Figure 5 is an SEM image of pre-osteogenic cells (MC3T3) cultured on bioglass sheets for various times.
  • Figure 6 is a graph showing changes in pH of an exemplary regenerative medical material sheet in a SBF solution of the present invention.
  • Figure 7 is a graph showing the change in weight of an exemplary regenerative medical material in water of the present invention.
  • Figure 8 is a graph showing the results of Regesi regenerative medicine applied to the repair of spinal and segmental bone defects.
  • Fig. 9 is a view showing the therapeutic effect of a repair material (gel) for ulcers developed based on Regesi regenerative medicine material.
  • Figure 10 is an experimental diagram of the easy application of Regesi regenerative medicine materials. It is indicated that the regenerative medicine material of the present invention can be melted into a film at body surface temperature.
  • Figure 11 is an experimental diagram of the effects of regenerative medical materials on wound healing. It shows that the regenerative medicine material of the present invention improves the speed and quality of wound healing, and has hair follicle formation.
  • Fig. 12 is a view showing regeneration of a bone defect site when a regenerative medicine material is used as a composite cancellous bone-filled porous stent. This description is similar to the mechanical properties of cancellous bone, and the material of the present invention promotes bone regeneration at the defect site.
  • Figure 13 is a comparison of the properties of the resulting bone when the regenerative medical material of the present invention is compared to the 45S material as a control. It shows that the degradation rate of 45S is too fast to form bone depression, and the hardness is too strong to cause peripheral fracture.
  • Figure 14 shows the effect of 45S extract on the proliferation of Hacat cells.
  • Figure 15 is a graph of the degradation experiment of the 45S powder.
  • regenerative medical material refers to an inorganic-organic composite material having a three-dimensional lattice structure that promotes soft and hard tissue regeneration functions, sometimes referred to as Regesi regenerative medical material, or Regesi et al., which are in the present invention.
  • the mass ratio of the inorganic substance to the organic substance in the regenerative medicine material is 2:1 to 4:1. If the mass ratio is less than 2:1, the hardness of the resulting regenerative medical material deteriorates and does not function well as a carrier for cell growth.
  • the mass ratio of the inorganic substance to the organic substance is from 2.5:1 to 3.8:1, more preferably from 2.6:1 to 3.5:1, further preferably from 2.8:1 to 3.4:1, for example, 3:1 or the like.
  • the SiO 2 content in the inorganic substance is 12.38%, preferably 15-35%, more preferably 16-33%, further preferably 18-30%, for example, 20%, 25, based on the total mass of the inorganic substance. %, 28%, 29%, etc.
  • the content of Na 2 O in the inorganic substance is 3-5%, preferably 3.5-4.5%, more preferably 3.6-4.2%, further preferably 4%.
  • the content of CaO in the inorganic substance is 15 to 29%, preferably 16 to 27%, more preferably 18 to 25%, still more preferably 20 to 22%.
  • the content of P 2 O 5 in the inorganic substance is from 10 to 32.5%, preferably from 12 to 30%, more preferably from 14 to 28%, further preferably from 16 to 26%, still more preferably from 18 to 24%, from 20 to 22%.
  • the content of phytate is 1-5%, preferably 2-4%, more preferably 3%.
  • the content of the cyclohexanol phosphate in the present invention is 1-5%, preferably 2-4%, more preferably 3%.
  • the content of each of the above components in the inorganic material is too low or too high, the content of each element in the regenerative medical material will not coincide with the content of elements in the body, such as a hard tissue such as bone in the human body, which is not conducive to promoting tissue regeneration or repair. .
  • the phytate and the cyclohexanol phosphate may be added as a separate component, or may be incorporated in the soybean extract to be mixed in the preparation process.
  • the inorganic substance is an impurity which is inevitably generated in the preparation process of the present invention except for the above components, and as an impurity, the content thereof is usually less than 0.5% by mass, preferably less than 0.4% by mass, more preferably less than 0.2% by mass, particularly It is preferably less than 0.1% by mass, most preferably 0.
  • the content of the carboxymethyl chitosan in the organic substance is from 30 to 60%, preferably from 40 to 55%, more preferably from 45 to 50%, further preferably 48%, based on the total mass of the organic substance.
  • the content of sodium hyaluronate in the organic substance is from 30 to 60%, preferably from 40 to 55%, more preferably from 45 to 50%, still more preferably 48%.
  • the weight ratio of calcium to phosphorus in the inorganic substance is from 1.5 to 1.8, preferably 1.67, and the ratio of calcium to phosphorus in the range corresponds to a ratio of calcium to phosphorus in a living body such as a human body.
  • the content of the components in the regenerative medicine material and the ratio between the components are prepared according to the content and ratio of each element in the hard tissue in the human body.
  • the content of each element in the hard tissue varies according to different human body, gender and different age groups. Therefore, the content and proportion of each component in the regenerative medicine material of the present invention may be different, but the content and ratio of the whole may not be Beyond the scope mentioned above.
  • the preferred ingredient content and ratio can be as follows:
  • the preferred calcium to phosphorus mass ratio is 1.67.
  • a specific plant extract solution is selected to achieve the beneficial technical effects of the regenerative medicine material of the present invention, for example, a low pH value as small as 7.4, P
  • the release of the plant is preferably controlled by a soybean extract.
  • the preparation method of the soybean extract comprises soybean smash pulverization ⁇ pulverization acid leaching, filtration ⁇ leaching liquid alkali neutralization ⁇ calcium salt precipitation leaching ⁇ RH + resin ion exchange ⁇ evaporation concentration ⁇ soybean extract, wherein The content of hydroxyphosphorus in the soybean extract needs to be 40 to 60% by weight.
  • the preparation steps of the soybean extract of the present invention are as follows:
  • the soluble calcium salt solution obtained by the dissolution is applied to an ion exchange column, and the flow rate is controlled for ion exchange.
  • impurity ions such as Mg 2+ and Ca 2+ in the solution are exchanged to the RH + resin, and the H + ions are After exchange, use about 1% of activated carbon to decolorize 1-2 times, separate, and then decolorize the liquid to evaporate and concentrate under reduced pressure, control temperature of about 70-80 ° C, until the solution in the bottle is thick, so that the soybean of the present invention
  • the extract has a hydroxyphosphorus content of 40 to 60% by weight.
  • the plant extract of the present invention is capable of producing the specific regenerative medicine material described in the present invention is not very clear, but it is speculated that it may be because the compatibility of the plant extract with the precursor of calcium (for example, calcium nitrate) is enhanced. So that no precipitation will occur.
  • the plant extract is a natural ingredient and is not toxic. Another reason may be that various other elements contained in the soybean extract interact with other materials in the present invention to produce an unexpected effect.
  • the content of each element in the soybean extract is similar to the composition of each element of the human body, and it is easy to obtain a composition similar to that of the body. Regenerative medical material.
  • the plant extract has a plurality of components capable of making the material have a three-dimensional structure, and a plurality of components interact with each other, thereby achieving the purpose of manufacturing a regenerative medical material at a low temperature.
  • the silicon precursor and the calcium precursor a precursor which is generally used in the art can be used. Further, water and/or ethanol may be used as the reaction medium in the production method of the present invention.
  • the gel precursor sol solution prepared by the above substances needs to be sintered at a relatively low temperature, for example, at a constant temperature of 300-700 ° C, and the temperature may preferably be 400-600 ° C, for example, 500 ° C, etc. temperature.
  • the "cell growth vector” refers to a substrate suitable for cells including, but not limited to, growth of bone cells, dermal cells, and proliferation.
  • the regenerative medical material of the invention itself can be used directly as a carrier.
  • the regenerative medical material of the present invention is combined/combined with other materials commonly used in the art as a carrier.
  • the corresponding content of the precursor was formulated into a gel precursor solution (the conversion of calcium nitrate tetrahydrate to calcium chloride or calcium nitrate did not affect the results).
  • tetraethyl orthosilicate (TEOS), ethanol and water (about 1:1 by volume) are added in order to dissolve the precursor.
  • TEOS tetraethyl orthosilicate
  • Ca(NO 3 ) 2 ⁇ 4H 2 O or calcium chloride or calcium nitrate
  • the prepared gel precursor sol solution is placed at room temperature until the gel (usually takes 2 to 10 days, depending on the ratio between the precursors), and the gel is aged in a 60 ° C oven for more than 1 day.
  • the carboxymethyl chitosan, sodium hyaluronate was mixed with the above powder formulation in proportion and heated to 45 degrees Celsius. Dissolve and stir well to obtain a mixture. 100 grams of medical glycerin is preheated to 80 degrees Celsius. The mixture was then incorporated into medical glycerin and stirred evenly (mass glycerol to biomaterial mass ratio of 55:45).
  • the Regesi regenerative medical material of the present invention was obtained by removing impurities, forming for 24 hours, and irradiating and sterilizing.
  • HA hydroxyapatite
  • the surface topography of the material was analyzed by scanning electron microscopy-energy dispersive x-ray spectroscopy (SEM-EDXS), as shown in Fig. 3.
  • SEM-EDXS scanning electron microscopy-energy dispersive x-ray spectroscopy
  • Fig. 3 The results show that the surface of the material is flat before deposition in SBF solution, and EDXS energy spectrum analysis proves that the main components are Si, P and Ca.
  • spherical particles appeared on the surface of the material. When the spherical particles were amplified, it was found that these particles were composed of acicular HA, and the content of Si and the content of Ca and P (Ca/P ⁇ 1.65) were decreased by EDXS energy spectrum analysis, which further confirmed the formation of HA.
  • 1% of Regesi regenerative medicine material powder was directly mixed with 10% DMEM/F12 medium, added to a 96-well plate, and then pre-osteogenic cells (MC3T3) were seeded at 96 ⁇ at 1 ⁇ 10 4 cells/mL.
  • DMEM/F12 medium was used as the control group.
  • MTT test was performed. After adding MTT, DMSO was added, and the absorbance was measured by enzyme-linked immunosorbent assay at 570 nm. (Absorbance), the measurement results are shown in Fig. 4 (a). The results indicate that Regesi regenerative medicine materials are not cytotoxic.
  • the absorbance of the material was slightly lower than that of the blank sample (about 92%), indicating that the Regesi regenerative medicine material was not cytotoxic.
  • the absorbance of the material was the same as that of the blank sample, indicating that Regesi regenerative medicine material No cytotoxicity.
  • the Regesi regenerative medicine material was immersed in 10% DMEM/F12 medium at a ratio of 5 mg/ml. After immersion for different time (1, 2, 3 d), the supernatant was centrifuged, and the 24-well plate extract was used as a blank control. The cells were stored at 4 °C, and the pre-osteogenic cells (MC3T3) were inoculated into 96-well plates at 1 ⁇ 10 4 cells/mL. After incubation in a CO2 incubator, extracts of different conditions were added for 24 hours (h). After the MTT test, MTT was added, DMSO was added, and the absorbance was measured by the enzyme-linked immunosorbent assay at a wavelength of 570 nm. The results are shown in Fig. 4(b). The results showed that the absorbance values of the extracts of Regesi regenerative medicine materials at different times were basically the same as those of the blank samples, indicating that the Regesi regenerative medicine material extract was still not cytotoxic.
  • the Regesi regenerative medicine material was ground into a powder, it was tableted (diameter 13 mm, thickness 2 mm).
  • the Regesi regenerative medicine material pieces were sterilized and disinfected, placed in a 24-well plate, and the pre-osteogenic cells (MC3T3) were inoculated into a 24-well plate at 1 ⁇ 10 4 /mL, and cultured for 1 d and 3 d. Then, it was fixed with 2.5% glutaraldehyde at 4 ° C for 24 h, washed 3 times with PBS, and washed with ethanol gradient (50%, 75%, 95% and 100%). After natural drying, gold was sprayed for SEM observation. As shown in Figure 5.
  • the Regesi regenerative medical material tablets (PSC, 45S5 and S70C30) were placed in a SBF solution to determine the pH change, and the results are shown in Fig. 6.
  • the pH values of the samples of 45S5 and S70C30 both reported products
  • the pH of the PSC sample the regenerative medicine material of the present invention
  • No change ⁇ 7.4, physiological pH
  • Previous studies have shown that the increase in pH is not conducive to cell growth.
  • the sample should be immersed in phosphate buffer solution for 24 hours to remove some ions on the surface of the sample.
  • PSC samples Prevent it from killing cells.
  • PSC samples generally do not require pretreatment due to their stable pH changes, and can directly perform cell compatibility experiments, and the above experimental results show that PSC has good cell compatibility, which is conducive to cell adhesion and proliferation. And differentiation. These results indicate that the PSC can be directly applied to the body after being sterilized and sterilized.
  • the Regesi regenerative medicine material piece was placed in deionized water (5 pieces / 50ml) for degradation test.
  • the deionized water was completely replaced at each interval, 5 pieces of sample were taken out, and the surface moisture was removed by paper. Dry in a vacuum dryer, weigh, and average, the results are shown in Figure 7. The results show that the material begins to degrade at 6d, accelerates degradation at 9d, and can degrade 40% in 70 days, and the degradation rate becomes slower.
  • the Regesi Regenerative Medicine material undergoes a degradation test in SBF solution to form HA on the surface of the material as the ions in the material are released. It will degrade to ⁇ 80% in the beginning of January, but after January, the surface of the material completely covers HA, resulting in little change in weight. Therefore, the degradation of Regesi regenerative medicine should be based on the degradation rate under its metabolism. quasi.
  • the degradation of the currently reported 45S5 bioglass is mainly investigated by implantation in vivo, and it takes about 1-2 years to completely degrade.
  • RegeSi bioactive glass was immersed in simulated body fluid to detect the change of pH value; biomechanical experiment was used to detect the maximum compressive strength in vitro; electron microscopic observation of the microstructure and surface of the material At the antennae, the surface of the material is sprayed with a gold film, and the surface topography and microporous structure of the material are scanned.
  • RegeSi bioactive glass was added into the osteoblast culture medium as the experimental group, and Gsk material was selected as the control group.
  • the effect of the material on the expression of type I collagen, osteocalcin and alkaline phosphatase gene in osteoblasts was detected by real-time quantitative PCR.
  • the cell and material composite test was carried out, and the materials of 1.0 cm ⁇ 1.0 cm ⁇ 0.5 cm were placed in the culture.
  • the cell suspension was inoculated on the surface of the material for composite culture, and the growth of the cells on the surface of the material was observed under an inverted phase contrast microscope.
  • the ulnar and lumbar vertebrae were examined by X-ray to observe the bone healing and defect repair.
  • 5 animals in 3 observation groups were killed, the titanium plates were removed surgically, and the adjacent vertebral bodies including the materials and the adjacent ulna at the defect were taken out.
  • the following studies were made: a. Making slices, microscopic observation of new filling parts of the material Bone growth and material degradation; b. Compressive and tensile strength of biomechanical test specimens; c. Specimen placed in Micro-CT system for 3D reconstruction observation, Microview ABA software for quantitative analysis of new bone minerals at bone graft Material content (TMC), bone volume fraction (BVF); d.
  • TMC bone graft Material content
  • BVF bone volume fraction
  • Overlay stacking method to display the residual materials and new bone in the bone graft area of each specimen in different colors.
  • the remaining 5 animals were surgically removed from the vertebral body under general anesthesia aseptic conditions.
  • the ulnar fixed titanium plate was used to suture the wound.
  • the movement and walking ability of the limbs of the sheep were observed after operation.
  • the animals were sacrificed 32 weeks after operation, and the restored vertebral body and ulna specimens were taken for biomechanical examination and histological analysis.
  • the Regei material of the present invention has excellent compressive strength, anti-stretching strength, and degradation as compared with the Gsk material group, the PMMA group, and the blank control group (see Fig. 8 and the like).
  • a 45S powder sample was used as the test object.
  • CCK-8 was used to measure the proliferative capacity of Hacat cells, and the absorbance-time curve was plotted.
  • the 10% concentration of the 45S extract was unstable, and a large amount of floc was precipitated during storage, and no subsequent tests were performed.
  • 1% of the extract was added to the next day and all cells died.
  • the 0.1% concentration of the extract had no significant cytotoxicity to the cells, but it can be seen from Fig. 14 that the cell growth was slowed compared to the control group at the end of the test.
  • the regenerative medicine material of the invention can be used for biological bone restoration toothpaste, tissue engineering regeneration, wound repair Re-medical materials, especially in tissue engineering, oral mucosa, bone repair materials, wound repair, etc. have great scientific value and significance. Will make important contributions in the field of tissue engineering and biotherapy.

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Abstract

一种促进软硬组织修复的再生医学材料及其制备方法和应用，再生医学材料具有三维网络结构，且为由无机物和有机物组成的复合材料，其中无机物与有机物的质量比为2:1-4:1。基于无机物的总质量，无机物中包含12-38％ SiO 2、3-5％ Na 2O、15-29％ CaO、10-32.5％ P 2O 5，1-5％肌醇六磷酸酯、1-5％环己六醇磷酸酯，余量为杂质，杂质的含量小于0.5％。基于有机物的总质量，有机物中包含30-60％羧甲基壳聚糖、30-60％透明质酸钠。该再生医学材料具有更加适于人体的组成及性质，在细胞修复和键合、细胞增殖、促进毛囊生长方面有关键作用。

Description

再生医学材料及其制备方法和应用 技术领域
本发明涉及再生医学领域，具体地涉及一种再生医学材料及其制备方法和应用。
背景技术
生物活性玻璃及玻璃陶瓷最显著的特征是植入人体后，表面状况随时间而动态变化，表面形成生物活性的碳酸羟基磷灰石(HCA)层，为组织提供了键合界面。多数生物活性玻璃是A类生物活性材料，既有骨生成性(osteoproductive)，又有骨引导(osteoconductive)作用，与骨和软组织都有良好的结合性，生物活性玻璃(BAG)被认为是可应用在修复领域的良好生物材料。此种修复性材料的用途不但极为广泛，而且在众多领域的专业性产品上有着无法替代的神奇功效，如肌肤护理、美白去皱、烧伤烫伤、口腔溃疡、肠胃溃疡、皮肤溃烂、杀灭真菌、骨骼修复、软组织和骨组织的键合等，它的出现将为人类健康做出卓越贡献。此外，BAG还具有快速的表面反应；无定形二维结构使强度及断裂韧性低；弹性模量(30-35MPa)低，与皮质骨接近；可切削生物玻璃具有良好的加工性能。
但是这种生物玻璃存在一些关键问题：1、降解速度缓慢，完全降解通常需要1-2年时间；2、pH值不稳定，其pH值可达到11，形成强碱性，具有一定的细胞毒性；3、这种生物玻璃采用熔融淬冷反应温度很高，在1700-1900℃，造成高能耗，建设一条标准生产线需要投资10亿元以上；4、这种生物玻璃不能形成多孔材料，难于实现材料高度符合及拓展功能。
因此，目前市场上急需能够代替上述生物玻璃的新型再生医学材料。
发明内容
为了解决上述技术问题，本发明的一方面，提供一种促进软硬组织修复的再生医学材料，所述再生医学材料具有三维网络结构，且为由无机物和有机物组成的复合材料，其中，所述无机物与所述有机物的质量比为2:1-4:1；
基于所述无机物的总质量，所述无机物包含12-38％SiO2、3-5％Na2O、15-29％CaO、10-32.5％P2O5，1-5％肌醇六磷酸酯、1-5％环己六醇磷酸酯，余量为杂质，其含量小于0.5％；
基于所述有机物的总质量，所述有机物包含30-60％羧甲基壳聚糖和30-60％透明质酸钠。
在优选的实施方案中，所述无机物与所述有机物的质量比为3:1。另外，所述无机物中的钙磷重量比为1.5至1.8，优选1.67。
在优选的实施方案中，所述无机物通过溶胶-凝胶法以大豆皮提取液作为磷前驱体，在300～700℃下恒温烧结得到。
本发明的另一方面，提供一种细胞生长载体，其包含上述促进软硬组织修复的再生医学材料。优选地，所述细胞生长载体为医疗装置，特别是植入物的至少一部分。
本发明的再一方面，提供一种促进软硬组织修复的方法，其中使用本发明中所述的再生医学材料。
本发明的其他方面，提供本发明所述的促进软硬组织修复的再生医学材料在制备医药组合物、医疗装置、口腔护理品、整形外科用品或化妆品中的用途。
本发明的再生医学材料能更好地控制材料的降解速度，使原生物活性玻璃降解速度可控，在人工骨材料应用领域使新生骨植入材料的降解速度同步，克服新生骨无法达到骨结构、形态、功能的三重修复的现象。当运用在骨修复、脊柱和骨缺损、口腔骨植入等方面时，本发明的再生医学材料具有非常重大的意义。
本发明中，通过大豆提取液引入磷，可以提高再生医学材料的降解速度， 传统的制备方法制备的材料的生物活性的组分范围较小，通常是通过降低磷的含量来限降解速度。原传统方法制备的材料通常用磷酸、磷酸乙酯等，其与钙的前驱体(例如硝酸钙)的相容性较差，容易引起沉淀发生分离。选择毒性较大的乙二醇，降低前驱体的浓度，这样处理溶剂过程中会耗费大量的能源和时间，并很难实现标准化、规模化生产。
本发明的磷前驱体可以有效地与例如硝酸钙等钙前驱体共溶，溶剂为水、乙醇或其混合物，毒性较小，溶剂去除温度较低，通过改变钙、钠等前驱体，实现室温状况下形成凝胶，实现在600℃内达到生物活性玻璃的各项理化指标。克服传统方法的缺点，实现标准化、规模化生产。
本发明的再生医学材料中，1％至10％的硅原子具有5以上的高配位数，并且这些高配位硅在常压下形成。高配位硅对材料的结构和性能都有一定的影响，如高配位硅会使紫外线吸收峰发生转移。
在溶液合成中，目前大多数高配位硅主要与N、F或CI配位，极少与O配位。通过改变外界刺激，如温度、溶剂和辐照强度可以改变硅原子的配位数，升高温度会使5配位的硅增加，在辐照下5配位硅与6配位硅之间可以相互转换。如果引入多元醇则有利于提高高配位硅的形成几率，多元醇能促使O与硅原子配位，从而形成高配位硅。在高配位硅，一般是在玻璃材料的高温处理，会使硅配位数由4转变为6，主要以Si-O-P或Si-O-Si的形式存在，随着P的含量增加，6配位硅增加，经过热处理，玻璃材料会结晶，而中低温、常压下含高配位硅原子的硅的固体材料目前研究还没有出现。
本发明的再生医学材料优于生物活性玻璃的理化指标及生物指标。该材料在生产时实现了在600℃低温节能，质量可控的规模化生产，改变原1700-1900℃煅烧高能耗、低产品合格率等缺点。在以该再生医学材料为母体的情况下，加入其它医用材料，将广泛的应用在生物治疗领域。
本发明的再生医学材料中，优选地，钙磷比与人体骨骼相对一致，例如为1.67左右。形成的三维网状结构及孔径可以实现和人体骨骼及软组织孔径一致。实现材料的生物活性、稳定性及降解速度可控。该材料制备温度低， 可以载入生物活性分子，例如蛋白质、抗菌素、化疗药物，并且具有多孔性，可以用于药物负载及控释，同时选用的磷前驱体为天然物质，与传统磷前驱体相对毒性极小，从而提高材料的生物相容性。材料在模拟人体液(SBF)中，在表面迅速形成羟基磷灰石。该材料可以克服传统生物活性玻璃的可塑性差。形成可调的无定形材料，非晶体结构。其他生物活性玻璃基本为结晶体，本发明的再生医学材料为非结晶体，材料的颗粒均匀、材料的生物相容性稳定，分散性较好，稳定性强。在人工骨中实现压缩强度、降解速度全部可控。
本发明的再生医学材料含有Si、Ca、Na、P离子，各离子的比例和人体骨组织的离子一致。这些离子均匀的分布在高温煅烧的SiO2的多孔径中，很自然使得材料与体液发生八步表面反应。
第一步：材料中的Na+和K+离子均匀分布在多孔径的SiO2中(其孔径和人体骨骼孔径几乎一致)，它们遇到体液，和H+以及H3O+离子迅速交换，如：
Si-O-Na++OH-→Si-OH++Na++OH-
其中OH-为负电荷，吸附骨组织及软组织细胞、生长因子、胶原蛋白等物质往孔径中有序生长，形成人体结构生长机块。让再生细胞基因表达，有序生长，形成骨引导、骨传导能力。
第二步：Si-O-Si键，被溶液打断，在界面外形成许多Si-OH；
第三步：Si-OH的聚合反应会形成SiO2的多孔的肢体层；其与不同种类的蛋白质通过氢键和离子胺键(-Si-O-H3N+-)结合形成高密度的蛋白吸附，形成硅溶胶层和碳酸羟基磷灰石层，而该灰石层具有高表面积，适合吸附大量的生物分子，从而促进细胞外响应。相比于带较低负电荷量的硅溶胶层，新生骨吸附更多的生物分子。
第四步：高配位硅原子6、硅原子4和人体钾离子交换，形成稳定的三维网状固态机构，改变原硅原子为游离状态。可与人体化学成分自然交换，形成爬行替代的载体，高配位硅会使紫外线吸收峰发生转移，起到抗氧化的作用。
Si-OH+OH-Si→Si-O-Si-+H2O；
第五步：Ca2+和PO4 3-来源材料中或来源与溶液中，在富SiO2肢体层上聚集形成CaO-P2O5无定型相层；通过P的比例，来调控新生细胞和原植入物的降解同步。
第六步：随着OH-和CO3 2-从溶液中引出，CaO-P2O5无定向相层将转变成含碳的羟基灰石(HCA)多晶体，吸附在创伤、溃疡及软组织表面，达到促进细胞增殖作用，促进创伤愈合、伤口无痕修复。
第七步：促进细胞增殖有序生长，促进毛囊的形成，促进毛囊的生长，达到减少疤痕的效果。
第八步：溃疡创面，特别是口腔溃疡，宫颈糜烂都是厌氧菌引起，本发明的再生医学材料能形成弱碱环境，让厌氧菌脱水而死，同时抑制厌氧菌的生长，促进新生细胞的修复，增殖。
综上所述，本发明的再生医学材料不仅具有适合于人体的各种性质，而且还具有与人体更加一致的成分及含量关系，从而更加适合于组织修复。
附图说明
图1为本发明示例性再生医学材料的X射线荧光光谱(XRF)的元素分布像(210×210μm2)。其表明本发明所述材料在微观尺度上的化学组成(Ca、P和Si)分布均一。
图2为本发明示例性再生医学材料与SBF溶液反应不同时间的XRD谱图。
图3为本发明的示例性再生医学材料的SEM-EDXS图。a-沉积前，b-SBF沉积后(14d)。
图4为本发明的示例性再生医学材料及其浸提液与成骨细胞相互作用的MTT值。(a)再生医学材料直接培养1d，6d；(b)Regesi再生医学材料不同时间的浸提液培养24h。
图5为成骨前细胞(MC3T3)在生物玻璃片上培养不同时间的SEM图。
图6为本发明的示例性再生医学材料片在SBF溶液中的pH值变化。
图7为本发明的示例性再生医学材料在水中的重量变化。
图8为Regesi再生医学材料应用于脊柱及节段骨缺损修复时的结果图。
图9为以Regesi再生医学材料为核心，研发的溃疡用修复材料(凝胶)的治疗效果的图。
图10为Regesi再生医学材料易于涂敷的实验图。其说明本发明的再生医学材料可在体表温度下融化成膜。
图11为再生医学材料对于伤口愈合影响的实验图。其说明本发明的再生医学材料提高了伤口愈合的速度和质量，有毛囊生成。
图12为再生医学材料作为复合松质骨填充多孔支架时，骨缺损部位再生的图。其说明与松质骨力学性能相似，本发明的材料促进缺损部位骨的再生。
图13为本发明的再生医学材料与作为对照的45S材料相比时，对于生成的骨的性能的比较。其说明45S降解速度过快易形成骨坍陷，硬度过强易引起周边骨折。
图14为45S浸提液对Hacat细胞增殖的影响。
图15为45S粉末的降解实验的图。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在 本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的材料和应用，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
本发明中，名词术语既包括单数形式，也包括复数形式，除非上下文另行明确指出。本发明中所述的“至少之一”或“至少一种”不仅仅指包含“一个”或“一种”的情况，更重要的还包含“多个”或“多种”的情况。
本发明所述的术语“再生医学材料”是指具有促进软硬组织再生功能的三维网格结构的无机-有机复合材料，有时还称作Regesi再生医学材料，或Regesi等，这些称号在本发明中具有相同的含义。优选地，再生医学材料中所述无机物与所述有机物的质量比为2:1-4:1。如果该质量比小于2:1，则所得再生医学材料的硬度变差，不能很好的作为细胞生长的载体。另一方面，如果该质量比大于4:1，则无机物中各元素的含量与人体组织，特别是硬组织例如骨骼中各元素的含量相差较大，从而不利于组织，特别是硬组织的再生。优选地，无机物与有机物的质量比为2.5:1-3.8:1，更优选2.6:1-3.5:1，进一步优选2.8:1至3.4:1，例如，3:1等。
本发明中，基于无机物的总质量，无机物中的SiO2含量为12-38％，优选15-35％，更优选16-33％，进一步优选18-30％，例如，20％、25％、28％、29％等。无机物中Na2O的含量为3-5％，优选为3.5-4.5％，更优选3.6-4.2％，进一步优选4％。无机物中CaO的含量为15-29％，优选为16-27％，更优选为18-25％，进一步优选为20-22％。无机物中P2O5的含量为10-32.5％，优选为12-30％，更优选14-28％，进一步优选16-26％，还优选18-24％，20-22％。本发明中，肌醇六磷酸酯的含量为1-5％，优选2-4％，更优选3％。本发明中环己六醇磷酸酯的含量为1-5％，优选2-4％，更优选3％。无机物中上述各成分的含量如果 过低或过高，则再生医学材料中各元素的含量与机体，例如人体中硬组织如骨骼的元素含量将不相一致，从而不利于促进组织再生或修复。
本发明中，作为肌醇六磷酸酯和环己六醇磷酸酯可作为单独的成分添加，也可通过包含于大豆提取液中而在制备过程中混入。
本发明中，无机物中除上述各成分外均为本发明制备过程中不可避免产生的杂质，作为杂质，其含量通常小于0.5质量％，优选小于0.4质量％，更优选小于0.2质量％，特别优选小于0.1质量％，最优选为0。
本发明中，基于有机物的总质量，有机物中羧甲基壳聚糖的含量为30-60％，优选40-55％，更优选45-50％，进一步优选48％。有机物中透明质酸钠的含量为30-60％，优选40-55％，更优选45-50％，进一步优选48％。
本发明中，优选地，所述无机物中的钙磷重量比为1.5至1.8，优选1.67，在该范围内的钙磷比符合机体如人体内的钙磷比。
需要说明的是，本发明中，再生医学材料中所成分的含量及各成分之间的比例是根据人体中硬组织中各元素的含量及比例配制而成。因不同人体、性别及不同年龄段，硬组织中各元素的含量有所不同，因此，本发明的再生医学材料中各成分的含量及比例也会有所不同，但整体上这些含量及比例不能超出上述提及的范围。
例如，对于老年人群体，其优选的成分含量及比例可如下所述：
Figure PCTCN2016096604-appb-000001
基于有机物的总质量。
对于老年人群体，优选的钙磷质量比为1.67。
对于年青人群体，其优选的成分含量及比例会适应调整。
本发明所述的促进软硬组织修复的再生医学材料的制备方法中，需选择特定的植物提取液，从而实现本发明再生医学材料所述有益技术效果，例如小至7.4的低pH值、P的释放可控，本发明所述的植物提取液优选为大豆提取液。在某些实施方案中，大豆提取液的制备方法包括大豆皮粉碎→粉碎料酸浸、过滤→浸出液碱中和→钙盐沉淀浸溶→RH+树脂离子交换→蒸发浓缩→大豆提取液，其中大豆提取液中羟基磷的含量需要为40-60重量％。
具体地，本发明的大豆提取液的制备步骤如下：
取粉碎后经筛分约20目的大豆皮，加6倍量的水。用7％盐酸，调pH至1.5～2，于室温搅拌浸渍。抽滤，用1.2％盐酸洗渣、弃渣，合并滤液。往浸出液中加石灰中和至pH值约6.5，得钙盐沉淀静止1h，抽滤，弃滤液再用蒸馏水洗涤所得沉淀物2-3次，得净化的钙盐。往所得钙盐中加少量稀盐酸并调成稀浆状，稍后加入2/3倍量H型强酸性阳离子交换树脂。微搅0.5h，使钙盐溶转成可溶性盐溶液。抽滤、洗净，分离，提取液粗液。
将溶转所得的可溶性钙盐溶液上柱到离子交换柱，控制流速进行离子交换.此时，溶液中的Mg2+、Ca2+等杂质离子被交换到RH+树脂上，H+离子被交换下来，用约1％量的活性炭脱色1-2次，分离，再将脱色液减压蒸发浓缩，控温70-80℃左右，至瓶内溶液呈稀稠状，即得本发明的大豆提取液，其羟基磷的含量需要为40-60重量％。
本发明所述的植物提取液能够产生得到本发明所述的特定再生医学材料的原因并不是非常清楚，但是推测可能是因为植物提取液与钙的前驱体(例如硝酸钙)的相容性增强，从而不会产生沉淀。另外，植物提取液为天然成分，没有毒性。另一原因可能在于，大豆提取液中含有的各种其他元素，与本发明中的其他原料相互作用产生了预期不到的效果。此外，大豆提取液中各元素的含量组成与人体各元素的组成相似，容易地获得与机体组成相似 的再生医学材料。
另外，植物提取液具有能够使材料具有三维结构的多种成分，并且多种成分之间相互作用，从而实现低温下制造再生医学材料的目的。克服传统方法的缺点，实现标准化、规模化生产。
本发明中作为硅前驱体、钙前躯体、可使用本领域内通常使用的前驱体。另外，本发明的制备方法中可以使用水和/或乙醇作为反应介质。本发明中，通过上述物质配制成的凝胶前驱体溶胶溶液需要在较低的温度下烧结，例如在300-700℃下恒温烧结，所述温度还可优选400-600℃，例如500℃等温度。
本发明中，所述的“细胞生长载体”是指适合细胞，包括但不限于骨细胞、真皮细胞生长、增殖的基质。优选地，本发明的再生医学材料本身可以直接作为载体。可选地，本发明的再生医学材料与本领域内通常使用的其他材料组合/复合后作为载体。
实施例
Regesi再生医学材料的制备方法
按照下述成分的含量，将相对应含量的前驱体配制成凝胶前驱体溶液(将四水硝酸钙换成氯化钙或硝酸钙均不影响结果)。
Figure PCTCN2016096604-appb-000002
首先，取本发明的大豆提取液30ml于50ml的样品瓶中，然后依次加入正硅酸乙酯(TEOS)，乙醇和水(体积比约为1∶1，加入量以能溶解前述前驱体即可)，搅拌30min，在搅拌过程中加入Ca(NO3)2·4H2O(或氯化钙、或硝酸钙)， 得到凝胶前驱体溶胶溶液。将配制的凝胶前驱体溶胶溶液在室温下放置直到凝胶(通常需要2～10天，取决于各前驱体之间的比例)，将凝胶放入60℃烘箱中陈化1天以上，然后再放入120℃烘箱中烘烤1周使其中的溶剂全部挥发，降温至室温。在空气中将管式炉的温度由室温以5℃/min的升温速度升温到300～400℃，将干的凝胶在300℃～400℃下的管式炉中恒温进行烧结至少10分钟后自然冷却，得到Regesi再生医学材料中的无机物粉体。
按比例将羧甲基壳聚糖、透明质酸钠与上述粉体配方混合，加热到45摄氏度。溶解，搅拌均匀，得到混合物。将100克医用甘油，预热到80摄氏度。之后将上述混合物融入医用甘油中，搅拌均匀(医用甘油与生物材料质量比为55∶45)。去除杂质，成化24小时，辐照灭菌，即得本发明的Regesi再生医学材料。
Regesi再生医学材料性质研究
1理化性质：
通过X射线荧光光谱(XRF)对材料的化学组成进行了分析，研究表明：材料在微观尺度上的化学组成(Ca、P和Si)分布均一，如图1所示。
将Regesi再生医学材料沉浸在模拟体液(SBF)中进行沉积实验，通过X射线衍射(XRD)研究发现，在沉积7天时，材料表面明显出现羟基磷灰石(HA)衍射峰(如图2所示)，而且随着时间的延长HA的衍射峰增强，说明材料表面有更多的羟基磷灰石形成。在沉积14天后，HA的衍射峰变化不大，说明形成的HA已经完全覆盖了材料表面。
通过扫描电镜-能量分散x射线能谱(SEM-EDXS)对材料的表面形貌进行分析，如图3所示。结果表明，在SBF溶液中沉积前，材料表面平整，EDXS能谱分析证明其主要组成是Si，P和Ca。而在SBF溶液中沉积14天后，材料表面出现球形粒子。将球形粒子放大后发现，这些粒子是由针状的HA组成，同时通过EDXS能谱分析发现Si含量减少，Ca、P含量(Ca/P～1.65)增加，进一步证明了HA的形成。这些结果与前面的XRD结果一致。
2生物学评价：
2.1.细胞毒性试验：
将1％的Regesi再生医学材料粉末直接与10％的DMEM/F12培养液共混，将其加入96孔板中，再将成骨前细胞(MC3T3)以1×104个/mL接种于96孔板内，DMEM/F12培养液作为对照组，CO2培养箱内分别培养1，6天(d)后，进行MTT试验，加入MTT后加入DMSO，震荡，酶联免疫法测量在570nm波长测定吸光度(Absorbance)，测定结果如图4(a)所示。结果表明Regesi再生医学材料不具有细胞毒性。在培养1d时，材料的吸光度稍微低于空白样品(约为其92％)，说明Regesi再生医学材料不具有细胞毒性，在培养6d时，材料的吸光度值与空白样品相同，说明Regesi再生医学材料无细胞毒性。
将Regesi再生医学材料以5mg/ml的比例浸入含10％的DMEM/F12培养液中，浸提不同时间后(1，2，3d)离心取上清液，24孔板浸提液作为空白对照组，均保存于4℃条件下，将成骨前细胞(MC3T3)以1×104个/mL接种于96孔板，CO2培养箱内培养后，加入不同条件的浸提液，24小时(h)后行MTT实验，加入MTT后加入DMSO，震荡，酶联免疫法测量在570nm波长测定吸光度，测定结果如图4(b)所示。结果表明Regesi再生医学材料不同时间的浸提液与空白样品的吸光度值基本相同，表明Regesi再生医学材料浸提液仍然没有细胞毒性。
2.2.细胞粘附性试验：
将Regesi再生医学材料研磨成粉末后，压片(直径13mm，厚度2mm)。将Regesi再生医学材料片进行灭菌、消毒处理，放入24孔板内，再将成骨前细胞(MC3T3)以1×104个/mL接种于24孔板内，培养1d和3d。然后用2.5％的戊二醛在4℃下固定24h，用PBS清洗3次，乙醇梯度脱洗(50％，75％，95％和100％)，经自然干燥后，喷金，进行SEM观察，如图5所示。结果表明，生物玻璃表面成骨细胞附着良好，培养1d时，细胞变长，有丝状伪足出现。培养3d时，细胞开始进一步延展，变大，伪足变得更为明显。这些证明Regesi再生医学材料具有良好的细胞相容性，有利于细胞在Regesi再生医学材料表 面附着。
3.降解实验：
将Regesi再生医学材料片(PSC、45S5和S70C30)放入SBF溶液中测定其pH值变化，结果如图6所示。从图中可以看出，45S5和S70C30(均为已报道产品)的样品pH值在初始的168h均升高，而PSC样品(本发明的再生医学材料)的pH值在这段时间内一直保持不变(～7.4，生理pH值))，如图6所示。通过前期研究表明，pH值升高不利于细胞的生长，一般对于45S5和S70C30样品的细胞相容性测试，均需将样品放入磷酸盐缓冲溶液中浸泡24h，除去样品表面的一些离子，以防止其杀死细胞。而PSC样品由于其稳定的pH值变化，一般不需要前处理，可直接进行细胞相容性实验，而且通过上面实验结果表明PSC具有很好的细胞相容性，有利于细胞的粘附、增殖及分化。这些结果表明PSC经消毒、灭菌后，可直接应用于体内。
将Regesi再生医学材料片放入去离子水中(5片/50ml)进行降解实验，在每个间隔时间点完全替换其中的去离子水，取出5片样品，用纸吸去其表面的水分，放入真空干燥器中烘干，称量，取平均值，结果如图7所示。结果表明：材料在6d开始降解，9d降解加快，70天降解能够达到原来的40％，且降解速率变缓。
Regesi再生医学材料在SBF溶液中进行降解试验，随着材料中离子的释放，在材料表面形成HA。在开始1月会降解为原来的～80％，但在1月后，材料表面完全覆盖成HA，导致其重量变化不大，所以Regesi再生医学材料的降解应以其体内新陈代谢下的降解速率为准。目前报道的45S5生物玻璃其降解主要是通过植入体内考察的，完全降解约需1-2年。
Regesi再生医学材料应用于脊柱及节段骨缺损修复的临床前研究
1、材料学
将RegeSi生物活性玻璃浸泡于模拟体液中，检测其pH值变化；生物力学实验检测其体外最大抗压缩强度；电子显微镜观察材料的显微形貌、表面接 触角，将材料表面喷镀金膜，扫描观察材料的表面形貌、微孔结构。
2、细胞学
在成骨细胞培养液中分别加入RegeSi生物活性玻璃作为实验组，选取Gsk材料作为对照组，利用实时定量PCR检测该材料对成骨细胞Ⅰ型胶原、骨钙素、碱性磷酸酶基因表达影响，以进一步探讨该材料促进成骨细胞增殖的可能机制；同时利用上述两种材料的提取液培养成骨细胞，MTT法测定细胞生长曲线，按6级毒性分级法计算相对增殖度RGR＝(实验组/对照组)×100％，并进行毒性评级；体外培养成骨细胞，然后分别接种至上述两种材料上，测定细胞粘附率和细胞粘附力，粘附率＝粘附细胞数/总细胞数×100％，用微吸管吸吮法测定系统测量细胞的粘附力，并进行扫描电镜观察；进行细胞与材料复合试验，分别取1.0cm×1.0cm×0.5cm大小的材料置于培养板内，将细胞悬液接种在材料表面进行复合培养，在倒置相差显微镜下观察细胞在材料表面的生长情况。
3、动物实验
1)选用雄性绵羊60只，平均体重75kg，分成3组，每组20只。在全麻无菌条件下行腰椎前路腰2椎体切除术，制作脊柱节段性骨缺损模型；同时制作尺骨中段4.5cm骨及骨膜缺损模型，将圆柱状RegeSi生物活性玻璃、Gsk材料、PMMA分别植入椎体切除后的间隙及尺骨缺损处，将三种修复材料分别用钛板固定在相邻节段的上下椎体及尺骨骨缺损修复处。在术后第1、6、12周行尺骨及腰椎正侧位X线检查观察骨愈合及缺损修复状况。同时分别处死3个观察组的5只动物，手术去除钛板，取出包括材料在内的相邻椎体及缺损处相邻尺骨，做以下研究：a.制作切片，显微镜观察材料填充部位的新骨生长以及材料降解情况；b.进行生物力学测试标本的抗压缩和牵拉强度；c.将标本置于Micro-CT系统进行三维重建观察，采用Microview ABA软件定量分析植骨处新生骨组织矿物质含量(TMC)、骨体积分数(BVF)；d.用Overlay叠加的方法用不同的颜色来复合显示各个标本植骨区域内的降解剩余材料和新生骨。于术后第24周将余下5只动物，在全麻无菌条件下手术取出椎体 及尺骨固定的钛板，缝合伤口，术后观察绵羊四肢的运动及行走能力，术后32周处死动物，取出修复后的椎体及尺骨标本做相应的生物力学检测及组织学分析。
2)将80只新西兰大白兔分为去势组和假手术组，去势组60只，假手术组20只，通过双侧卵巢去势法建立兔骨质疏松模型。将去势组分为Regei组、PMMA组、空白对照组，每组20只，模拟椎体成形术，分别在Regei组兔子L1、L2椎体注射可注射的RegeSi生物活性玻璃，在PMMA组兔子L1、L2椎体注射PMMA，每组均于术后第1、6、12、24周处死5只兔子，取出相应的椎体，采用组织学、MicroCT分析、显微镜填充部位的新骨生长以及材料降解情况，利用生物力学实验评估Regei生物活性玻璃抗压强度、抗牵拉强度。
通过上述实验，证实本发明的Regei材料与Gsk材料组、PMMA组、空白对照组相比，具有优异的抗压强度、抗牵拉强度以及降解情况等(参见图8等)。
现有生物活性玻璃的性质研究
以45S粉末状样品作为测试对象。
实验过程：
1)45S高温高压灭菌。
2)浸提液制备：将灭菌后的45S浸泡在相应体积的MEM培养基中，37oC浸泡24小时。质量体积百分比分别是0.1％，1％，10％。
3)浸提液取上清，用1M HCl将pH调至7.2。0.22μm滤头灭菌。4℃保存。
4)三种浓度浸提液分别加入10％FBS。
5)CCK-8测Hacat细胞的增殖能力，绘制吸光度-时间曲线。
测试结果：
10％浓度的45S浸提液不稳定，存放过程中会析出大量絮状物，未进行后续测试。CCK-8测试过程中发现1％的浸提液加入第二天细胞全部死亡。 0.1％浓度的浸提液对细胞没有明显的细胞毒性，但是从图14可知在测试后期细胞生长相比对照组减慢。
补充实验(45S浸泡于蒸馏水中对pH值的影响)
实验过程：将45S按照质量体积比1％的浓度浸泡于蒸馏水中，测试不同时间点的pH值。
测试结果：
Figure PCTCN2016096604-appb-000003
结论：
结果显示45S浸提液随着浸泡时间的增加不断升高，24小时pH值内达13。pH大幅升高导致CCK-8测试中显示很强的细胞毒性。
其他实验
1)将高温高压灭菌后的45S加入到MEM培养基中。45S的体积百分比分别为0.1％，1％，10％。45S一加入MEM培养基中就有明显的颜色变化，说明培养基的pH变化较大。浸泡24h后，取上清用1M HCl调节pH至7.2。随着45S浓度的增加，培养基碱性越强，所需HCl量越大。为确认实验结果，测试了45S在蒸馏水中浸泡后的pH值变化。测试结果仍然显示45S引起pH大幅度增加，产生细胞毒性。
2)从4℃冰箱取出准备好的45S浸提液发现10％浓度样品中有大量白色絮状物。37℃水浴加热半小时不溶解。此浓度样品没有进行后续CCK-8测试。室温静置两周后10％浓度的45S粉末在MEM中呈溶胶状。说明45S粉末的降解速度快，参见图15。
3)用0.1％和1％浓度浸提液加FBS进行CCK-8测试过程中，发现1％浓度的浸提液有很大的细胞毒性。细胞全部死亡。0.1％浓度的浸提液有较小细胞毒性。
工业实用性
本发明的再生医学材料可用于生物骨修复牙膏、组织工程再生、创伤修 复医用材料，特别是在：组织工程、口腔粘膜、骨修复材料、创伤修复等方面有着重大的科学价值和意义。将在组织工程、生物治疗领域做出重要贡献。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本申请说明书和实施例仅是示例性的。

Claims (11)

  1. 一种促进软硬组织修复的再生医学材料，所述再生医学材料具有三维网络结构，且为由无机物和有机物组成的复合材料，其中，所述无机物与所述有机物的质量比为2:1-4:1；
    基于所述无机物的总质量，所述无机物包含12-38％SiO2、3-5％Na2O、15-29％CaO、10-32.5％P2O5，1-5％肌醇六磷酸酯、1-5％环己六醇磷酸酯，余量为杂质，所述杂质的含量小于0.5％；
    基于所述有机物的总质量，所述有机物包含30-60％羧甲基壳聚糖、30-60％透明质酸钠。
  2. 根据权利要求1所述的促进软硬组织修复的再生医学材料，其中，所述无机物与所述有机物的质量比为3:1。
  3. 根据权利要求1或2所述的促进软硬组织修复的再生医学材料，其中，所述无机物中的钙磷重量比为1.50至1.80。
  4. 根据权利要求1或2所述的促进软硬组织修复的再生医学材料，其中，所述无机物中的钙磷重量比为1.67。
  5. 根据权利要求1或2所述的促进软硬组织修复的再生医学材料，其pH值为7.4±1。
  6. 根据要求1或2所述的促进软硬组织修复的再生医学材料，其在体内的降解速率为4周至12周。
  7. 根据权利要求1至6任一项所述的促进软硬组织修复的再生医学材料的制备方法，其包括以下步骤：
    以大豆提取液作为磷前驱体，以正硅酸乙酯作为硅前驱体，以选自四水硝酸钙、硝酸钙和氯化钙的至少之一作为钙前躯体，以水和/或乙醇作为反应介质，将上述各前躯体及反应介质混合配制成凝胶前驱体溶胶溶液，在300～700℃下恒温烧结得到无机物；
    将所述无机物与羧甲基壳聚糖、透明质酸钠混合，加热溶解。
  8. 根据权利要求7所述的方法，其中所述大豆提取液的制备方法包括：大豆皮粉碎→粉碎料酸浸、过滤→浸出液碱中和→钙盐沉淀浸溶→RH+树脂 离子交换→蒸发浓缩→大豆提取液，其中大豆提取液中羟基磷的含量为40-60重量％。
  9. 一种细胞生长载体，其包含根据权利要求1至6任一项所述的促进软硬组织修复的再生医学材料。
  10. 一种促进软硬组织修复的方法，其使用根据权利要求1至6任一项所述的再生医学材料。
  11. 根据权利要求1至6任一项所述的再生医学材料在制备医药组合物、医疗装置、口腔护理品、整形外科用品或化妆品中的用途。
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