CN110101917B - Calcified layer-containing cartilage scaffold with slow-release double growth factors and preparation method thereof - Google Patents
Calcified layer-containing cartilage scaffold with slow-release double growth factors and preparation method thereof Download PDFInfo
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Abstract
The invention discloses a calcified layer containing osteochondral scaffold capable of slowly releasing double growth factors and a preparation method thereof. The constructed osteochondral scaffold has a structure similar to that of natural cartilage, and comprises an upper oriented structure layer, a compact calcified intermediate layer and a reticular porous lower layer, wherein polyester material microspheres for slowly releasing transforming growth factors are introduced into the upper layer of scaffold, the lower layer of scaffold comprises polyester material microspheres for slowly releasing bone morphogenetic protein growth factors, and the stabilization of cartilage and bone microenvironment and the function release of different areas of the scaffold are promoted through material structure design, so that the constructed osteochondral scaffold has a good effect of promoting the repair and regeneration of osteochondral.
Description
Technical Field
The invention relates to a cartilage scaffold containing calcified layer and sustained-release double-growth factor and a preparation method thereof, in particular to a cartilage scaffold containing calcified layer and a preparation method thereof, wherein growth factor-loaded polyester material microspheres are loaded on a fibroin composite material to form the cartilage scaffold. Belongs to the field of biological material and biomedical engineering.
Background
The joint injury of bone and cartilage caused by trauma, tumor excision, osteoarthritis and the like is a common disease in orthopedics. The existing treatment method for the osteochondral combined injury comprises autologous or allogeneic osteochondral transplantation, but has the problems of donor area injury or immunological rejection, and the overall treatment effect is poor, so that breakthrough progress is not achieved. The tissue engineering technology provides a new idea for treating osteochondral defects.
The process of osteochondral regeneration involves the regeneration of cartilage and bone. Because stem cells have the potential of multidirectional differentiation, how to induce stem cells to directionally induce and differentiate into specific cells at specific positions of a scaffold in tissue engineering is very important, and in recent years, development of a bioactive scaffold system loaded with growth factors is widely concerned. In the in situ regeneration of articular cartilage, Transforming Growth Factors (TGFs) are proteins that affect cell growth, proliferation and differentiation. TGFs mainly include two types, TGF-alpha and TGF-beta, respectively. Both types have unique amino acid sequences and modes of action that differ from the receptor. TGF-. beta.s have been shown to affect differentiation of stem cells into cartilage. Furthermore, Bone Morphogenic Proteins (BMPs) were the first active substances found for bone regeneration. BMPs can induce the formation of ectopic bone and cartilage. At present, a total of 20 bone morphogenetic proteins have been discovered, including BMP-1 through BMP-18, BMP-3b, and BMP-8 b. Among them, BMP-2 is a bone morphogenetic protein which is most widely used and promotes osteogenic differentiation and bone formation of stem cells. However, TGFs and BMPs have the problems of relatively short action time, easy degradation and the like, and if the release can be controlled slowly, the biological effect can be better exerted.
Actually, cartilage and bone tissues are in two independent and different microenvironments, cartilage is in a low-oxygen and blood-supply-free environment, subchondral bone is in a high-oxygen and rich blood-supply environment, and the cartilage and the bone are isolated by a calcification layer (CCL) which plays a role of a barrier. If the CCL layer is lacked in the regenerated osteochondral tissue, the CCL barrier function disappears, the microenvironment of cartilage and bone is disturbed, the regeneration of cartilage is influenced, and the long-term repair effect of osteochondral is finally influenced.
Disclosure of Invention
Aiming at the problems of rapid inactivation of growth factors and disorder of microenvironment of cartilage and bone in the existing osteochondral scaffold material, the invention prepares the calcified layer osteochondral scaffold containing the slow-release double growth factors, so that the cartilage scaffold can keep the activity of the growth factors and the stability of the microenvironment of the cartilage and the bone and promote the regeneration of the osteochondral.
The cartilage scaffold containing calcified layer of the slow-release double-growth factor is similar to a natural osteochondral structure and comprises an oriented upper layer, a calcified middle layer and a reticular porous lower layer, wherein the oriented upper layer is composed of silk fibroin, acellular cartilage extracellular matrix and polyester material microspheres of the slow-release TGF-beta growth factor; the calcification intermediate layer is composed of silk fibroin and nano hydroxyapatite; the lower reticular porous layer is composed of silk fibroin, nano hydroxyapatite and polyester material microspheres for slowly releasing BMPs growth factors. Through structural design and growth factor controlled release design, the stabilization of the micro environment of cartilage and bone and the slow release of TGF-beta and BMPs dual factors are realized, and the aim of effectively repairing cartilage defects can be achieved. The specific preparation method comprises the following steps:
1) preparing growth factor-loaded polyester microspheres: firstly, 50-200mg of polyester material is dissolved in 1mL of dichloromethane to be used as an oil phase (O), 1mL of aqueous solution containing 5-10 mu g of BMPs or TGF-beta (internal water phase W1) is added, a homogenizing emulsifier is used for preparing colostrum (W1/O), then the colostrum is added into 10-50mL of aqueous solution (W2) containing 0.5-3% PVA, and the homogenizing emulsifier is used for stirring to prepare multiple emulsion (W1/O/W2); and magnetically stirring the double emulsion at 600rpm for 3-6h to solidify the microspheres, centrifugally washing the solidified microspheres for 3 times by using distilled water, freezing the microspheres in a freezer at the temperature of-80 ℃, and finally, freeze-drying for 48h to obtain the growth factor-loaded polyester microspheres.
2) Preparing a reticular porous lower-layer scaffold: firstly, weighing silk fibroin and nano hydroxyapatite solid according to the mass ratio of 1: 1, dissolving the solid in hexafluoroisopropanol to prepare a mixed solution with the concentration of 4-30%, injecting the mixed solution and sodium chloride crystal particles into a cylindrical die with the diameter of 4-16mm according to the volume ratio of 1: 2, placing and drying the silk fibroin at room temperature for 48h, soaking the silk fibroin in anhydrous alcohol for 0.5h, and transferring the silk fibroin into distilled water to soak the silk fibroin to remove the sodium chloride particles. After freeze drying, preparing microsphere suspension aqueous solution according to the mass ratio of the microspheres to the scaffold of 0.15: 10-1.2: 10, sucking the microsphere suspension into the scaffold under negative pressure, and freeze-drying the scaffold for 48h for the second time to obtain a lower-layer scaffold containing sustained-release BMPs growth factor microspheres;
3) preparing a calcified middle layer-reticular porous lower layer scaffold: dripping 0.1-0.5mL of 10-30% mixed solution of nano-hydroxyapatite and silk fibroin hexafluoroisopropanol with the mass ratio of 1: 1 on the surface of the reticular porous lower-layer stent prepared in the step 2), and drying at room temperature to obtain a calcified middle layer and a calcified lower-layer stent;
4) preparing an upper layer-calcified middle layer-reticular porous lower layer scaffold: preparing a composite solution with the total concentration of 3% -6% according to the mass ratio of 1: 1 of the decellularized cartilage extracellular matrix to the silk fibroin, fully mixing, adding microspheres carrying growth factors accounting for 24% -192% of the mass of the decellularized cartilage extracellular matrix, uniformly stirring, then adding 0.5-2mL of the composite solution containing the microspheres to the surface of the calcification layer bracket prepared in the step 3), rapidly carrying out gradient cooling at room temperature to-20 ℃, and preparing the oriented decellularized cartilage extracellular matrix and silk fibroin orientation upper layer bracket; and finally, freeze-drying the integral scaffold, performing ethanol treatment, removing ethanol, and performing secondary integral freeze-drying for 48 hours to obtain the calcified layer cartilage scaffold with the function of slowly releasing the double growth factors.
The polyester material comprises polylactic-co-glycolic acid (PLGA) and polylactic acid (PLA); the growth factor comprises transforming growth factor beta1(TGF-β1) Transforming growth factor beta2(TGF-β2) Transforming growth factor beta3(TGF-β3) Bone morphogenetic protein 2(BMP-2) and bone morphogenetic protein 7 (BMP-7).
Compared with the prior art, the invention has the outstanding advantages that:
1) in the selection of the material, the biodegradable natural silk fibroin has better biocompatibility and mechanical property, so that the material meets the requirements of the osteochondral scaffold; the nano hydroxyapatite is an important component of human osteogenesis and has osteogenesis guiding property in the process of bone regeneration; the decellularized chondrocyte extracellular matrix also has good biocompatibility, and simultaneously has the effect of promoting the differentiation of mesenchymal stem cells to chondrocytes.
2) In the preparation process, the microspheres are prepared by an emulsification-solvent evaporation method, and the osteochondral scaffold is prepared by a particle leaching technology and a thermally induced phase separation technology. The emulsification-solvent evaporation method can be used for loading various water-soluble medicines, and has simple preparation method and high medicine loading rate; the particle leaching technology can obtain the scaffold material with different apertures by controlling the size of particles, the operation is simple, and the aperture of the scaffold is controllable; the thermally induced phase separation technology can realize the oriented structures with different pore sizes by controlling the crystallization temperature, and has simple method and mild and controllable preparation conditions.
3) Functionally, the porous material prepared by adopting the particle leaching technology and the thermally induced phase separation technology has the characteristic of high structural bionics, the existence of the calcified layer is favorable for preventing the cartilage cells and the osteoblasts from migrating and infiltrating with each other, an independent microenvironment is provided for the repair and regeneration of the cartilage and the osteogenesis, the biological activity of the growth factors is improved by adding the slow-release microsphere material, and the function of inducing the stem cell differentiation is given to the scaffold material, so that the regeneration of the bone and the cartilage is favorable.
Example 1:
1) 50mg PLGA was dissolved in 1mL of dichloromethane as oil phase (O), and 0.1mL of 5. mu.g BMP-2 or TGF-. beta.was added3An aqueous solution of growth factors (inner aqueous phase W1), colostrum (W1/O) was prepared using a homogenizer, and then colostrum was added to 30mL of an aqueous solution (W2) containing 0.5% PVA to prepare double emulsion (W1/O/W2) with continuous stirring using a homogenizer. And magnetically stirring the double emulsion at 600rpm for 3h to solidify the microspheres, centrifugally washing the solidified microspheres for 3 times by using distilled water, freezing the microspheres in a freezer at the temperature of-80 ℃, and freeze-drying the microspheres for 48h to obtain the PLGA microspheres carrying the growth factors.
2) Weighing silk fibroin and nano hydroxyapatite solid according to the mass ratio of 1: 1, dissolving the solid in hexafluoroisopropanol to prepare a mixed solution with the concentration of 8%, injecting 1mL of the mixed solution into a cylindrical mold containing 2g of sodium chloride crystal particles, standing and drying the mixed solution at room temperature for 48h, soaking the mixed solution in anhydrous alcohol for 0.5h, and transferring the mixed solution to distilled water to soak the mixed solution to remove the sodium chloride particles. After freeze drying, preparing microsphere suspension aqueous solution according to the mass ratio of the microspheres to the scaffold of 0.15: 10, putting the scaffold into the microsphere suspension aqueous solution, sucking the microsphere suspension into the scaffold under negative pressure, and freeze-drying the scaffold for 48h for the second time to obtain a lower-layer scaffold containing sustained-release BMP-2 growth factor microspheres;
3) dripping 0.2mL of 10% mixed solution of silk fibroin and nano-hydroxyapatite hexafluoroisopropanol at a mass ratio of 1: 1 on the surface of the lower-layer bracket, and drying at room temperature to obtain a calcified layer and the lower-layer bracket;
4) preparing a composite solution with the total concentration of 3% according to the mass ratio of the decellularized cartilage extracellular matrix to the silk fibroin of 1: 1, fully mixing, and adding a solution containing TGF-beta accounting for 24% of the weight of the decellularized cartilage extracellular matrix3Microspheres are uniformly stirred, 1mL of composite solution containing the microspheres is added to the surface of the calcified layer bracket, gradient cooling is rapidly carried out at minus 20 ℃, and the cartilage extracellular matrix oriented to decellularize and the silk fibroin oriented upper layer bracket are prepared. And finally, freeze-drying the integral scaffold, performing ethanol treatment, removing ethanol, and performing secondary integral freeze-drying for 48 hours to obtain the calcified layer cartilage scaffold with the function of slowly releasing the double growth factors.
Example 2:
1) 100mg PLGA was dissolved in 1mL of dichloromethane as oil phase (O), and 0.1mL of 5. mu.g BMP-2 or TGF-. beta.was added3An aqueous solution of growth factors (inner water phase W1), colostrum (W1/O) was prepared using a homogenizer, and then colostrum was added to 40mL of an aqueous solution (W2) containing 1% PVA to prepare double emulsions (W1/O/W2) with stirring using a homogenizer. And magnetically stirring the double emulsion at 600rpm for 4h to solidify the microspheres, centrifugally washing the solidified microspheres for 3 times by using distilled water, freezing the microspheres in a freezer at the temperature of-80 ℃, and freeze-drying the microspheres for 48h to obtain the PLGA microspheres carrying the growth factors.
2) Weighing silk fibroin and nano hydroxyapatite solid according to the mass ratio of 1: 1, dissolving the solid in hexafluoroisopropanol to prepare a mixed solution with the concentration of 8%, injecting 1mL of the mixed solution into a cylindrical mold containing 2g of sodium chloride crystal particles, standing and drying the mixed solution at room temperature for 48h, soaking the mixed solution in anhydrous alcohol for 0.5h, and transferring the mixed solution to distilled water to soak the mixed solution to remove the sodium chloride particles. After freeze drying, preparing microsphere suspension aqueous solution according to the mass ratio of the microspheres to the scaffold of 0.3: 10, putting the scaffold into the microsphere suspension aqueous solution, sucking the microsphere suspension into the scaffold under negative pressure, and performing secondary freeze drying on the scaffold for 48 hours to obtain a lower-layer scaffold containing the slow-release BMP-2 growth factor microspheres;
3) dripping 0.5mL of mixed solution of 12% silk fibroin and nano hydroxyapatite hexafluoroisopropanol at a mass ratio of 1: 1 on the surface of the lower layer bracket, and drying at room temperature to obtain a calcified layer and the lower layer bracket;
4) preparing a composite solution with the total concentration of 4% according to the mass ratio of the decellularized cartilage extracellular matrix to the silk fibroin of 1: 1, fully mixing, and adding a solution containing TGF-beta accounting for 48% of the weight of the decellularized cartilage extracellular matrix3And uniformly stirring the microspheres, adding 1mL of microsphere composite solution to the surface of the calcified layer support, and rapidly performing gradient cooling at-20 ℃ to prepare the cartilage extracellular matrix oriented to decellularize and the silk fibroin oriented upper layer support. And finally, freeze-drying the integral support, then carrying out ethanol treatment, carrying out secondary integral freeze-drying for 48 hours after ethanol removal, and thus obtaining the calcified layer containing cartilage support with the function of slowly releasing the double growth factors.
Example 3:
1) 200mg PLGA was dissolved in 1mL of dichloromethane as oil phase (O), and 0.1mL of 5. mu.g BMP-2 or TGF-. beta.was added3An aqueous solution of growth factors (inner water phase W1), colostrum (W1/O) was prepared using a homogenizer, and then colostrum was added to 50mL of an aqueous solution (W2) containing 2% PVA to prepare double emulsions (W1/O/W2) with stirring using a homogenizer. And magnetically stirring the double emulsion at 600rpm for 4h to solidify the microspheres, centrifugally washing the solidified microspheres for 3 times by using distilled water, freezing the microspheres in a freezer at the temperature of-80 ℃, and freeze-drying the microspheres for 48h to obtain the PLGA microspheres carrying the growth factors.
2) Weighing silk fibroin and nano hydroxyapatite solid according to the mass ratio of 1: 1, dissolving the solid in hexafluoroisopropanol to prepare a mixed solution with the concentration of 8%, injecting 1mL of the mixed solution into a cylindrical mold containing 2g of sodium chloride crystal particles, standing and drying the mixed solution at room temperature for 48h, soaking the mixed solution in anhydrous alcohol for 0.5h, and transferring the mixed solution to distilled water to soak the mixed solution to remove the sodium chloride particles. After freeze drying, preparing microsphere suspension aqueous solution according to the mass ratio of the microspheres to the scaffold of 1.2: 10, putting the scaffold into the microsphere suspension aqueous solution, sucking the microsphere suspension into the scaffold under negative pressure, and freeze-drying the scaffold for 48h for the second time to obtain a lower-layer scaffold containing sustained-release BMP-2 growth factor microspheres;
3) dripping 0.5mL of mixed solution of 16% silk fibroin and nano hydroxyapatite hexafluoroisopropanol at a mass ratio of 1: 1 on the surface of the lower layer bracket, and drying at room temperature to obtain a calcified layer and the lower layer bracket;
4) preparing a composite solution with the total concentration of 3% according to the mass ratio of the decellularized cartilage extracellular matrix to the silk fibroin of 1: 1, fully mixing, and adding a solution containing TGF-beta accounting for 192% of the weight of the decellularized cartilage extracellular matrix3And (3) uniformly stirring microspheres, adding 1mL of microsphere composite solution to the surface of the calcified layer support, and rapidly performing gradient cooling at-20 ℃ to prepare the oriented acellular cartilage extracellular matrix and silk fibroin oriented upper layer support. And finally, freeze-drying the integral scaffold, performing ethanol treatment, removing ethanol, and performing secondary integral freeze-drying for 48 hours to obtain the calcified layer cartilage scaffold with the function of slowly releasing the double growth factors.
Claims (1)
1. The calcified layer osteochondral scaffold containing the slow-release double-growth factor is similar to a natural osteochondral structure and comprises an oriented upper layer, a calcified middle layer and a reticular porous lower layer, wherein the oriented upper layer is composed of silk fibroin, acellular cartilage extracellular matrix and polyester material microspheres of the slow-release TGF-beta growth factor; the calcification intermediate layer is composed of silk fibroin and nano hydroxyapatite; the lower reticular porous layer is composed of silk fibroin, nano-hydroxyapatite and polyester material microspheres for slowly releasing BMPs growth factors;
the preparation method of the calcified layer containing osteochondral scaffold with the slow-release double growth factors comprises the following steps:
1) preparing growth factor-loaded polyester microspheres: firstly, 50-200mg of polyester material is dissolved in 1mL of dichloromethane to be used as an oil phase (O), 1mL of aqueous solution containing 5-10 mu g of BMPs or TGF-beta (internal water phase W1) is added, a homogenizing emulsifier is used for preparing colostrum (W1/O), then the colostrum is added into 10-50mL of aqueous solution (W2) containing 0.5-3% PVA, and the homogenizing emulsifier is used for stirring to prepare multiple emulsion (W1/O/W2); magnetically stirring the double emulsion at 600rpm for 3-6h to solidify the microspheres, centrifugally washing the solidified microspheres for 3 times by using distilled water, freezing the microspheres in a freezer at-80 ℃, and finally, freeze-drying for 48h to obtain the polyester microspheres loaded with the growth factors;
2) preparing a reticular porous lower-layer scaffold: firstly, weighing silk fibroin and nano hydroxyapatite solids according to the mass ratio of 1: 1, dissolving the silk fibroin and nano hydroxyapatite solids in hexafluoroisopropanol to prepare a mixed solution with the concentration of 4% -30%, injecting the mixed solution and sodium chloride crystal particles into a cylindrical die with the diameter of 4-16mm according to the volume ratio of 1: 2, placing and drying the cylindrical die at room temperature for 48 hours, soaking the cylindrical die with anhydrous alcohol for 0.5 hour, and transferring the cylindrical die into distilled water to soak the cylindrical die to remove sodium chloride particles; after freeze drying, preparing microsphere suspension aqueous solution according to the mass ratio of the microspheres to the scaffold of 0.15: 10-1.2: 10, sucking the microsphere suspension into the scaffold under negative pressure, and freeze-drying the scaffold for 48h for the second time to obtain a lower-layer scaffold containing sustained-release BMPs growth factor microspheres;
3) preparing a calcified middle layer-reticular porous lower layer scaffold: dripping 0.1-0.5mL of 10-30% mixed solution of nano hydroxyapatite and silk fibroin hexafluoroisopropanol with the mass ratio of 1: 1 on the surface of the reticular porous lower layer bracket prepared in the step 2), and drying at room temperature to obtain a calcified middle layer and a calcified lower layer bracket;
4) preparing an upper layer-calcified middle layer-reticular porous lower layer scaffold: preparing a composite solution with the total concentration of 3-6% according to the mass ratio of the decellularized cartilage extracellular matrix to the silk fibroin of 1: 1, fully mixing, adding microspheres carrying growth factors accounting for 24-192% of the mass of the decellularized cartilage extracellular matrix, uniformly stirring, then adding 0.5-2mL of the composite solution containing the microspheres to the surface of the calcified layer bracket prepared in the step 3), rapidly performing gradient cooling at room temperature to-20 ℃, and preparing an oriented decellularized cartilage extracellular matrix and silk fibroin oriented upper layer bracket; and finally, freeze-drying the integral scaffold, performing ethanol treatment, removing ethanol, and performing secondary integral freeze-drying for 48 hours to obtain the calcified layer cartilage scaffold with the function of slowly releasing the double growth factors.
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| CN110433331B (en) * | 2019-08-26 | 2021-08-24 | 四川大学 | Bioactive scaffold and preparation method thereof |
| CN110772668B (en) * | 2019-09-30 | 2021-02-19 | 北京化工大学 | Soft and hard tissue integrated regeneration and repair gradient three-dimensional fiber scaffold and preparation method thereof |
| TWI723615B (en) * | 2019-11-04 | 2021-04-01 | 中國醫藥大學 | Biological scaffold for promoting bone tissue repair, use thereof, and method for preparing the same |
| WO2023115295A1 (en) * | 2021-12-21 | 2023-06-29 | 浙江大学 | Multi-layer composite material for promoting articular cartilage-calcified layer-subchondral bone repair and preparation method therefor |
| CN114949371B (en) * | 2022-04-12 | 2023-04-14 | 重庆大学 | Double-layer porous scaffold for repairing articular cartilage defect and preparation method thereof |
| CN115607742A (en) * | 2022-10-26 | 2023-01-17 | 兰州大学 | Composite hydrogel scaffold for promoting bone-cartilage repair and preparation method and application thereof |
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