TWI440486B - A polylactic acid/calcium sulfate scaffold - Google Patents

Description

Polylactic acid/calcium sulfate stent

The present invention relates to a method of preparing a composite or porous composite for use as a biodegradable stent, and the composites thereof and the use of the composite. In particular, the composite is a calcium sulphate (CS)-polylactic acid (PLA) composite or a porous composite, and the composite is particularly useful as a scaffold for in situ pore formation.

Bone defects in fractures, hereditary malformations, tumors, and spinal surgery often require implants to be implanted. A typical absorbable tissue engineering scaffold should have sufficient porosity to allow osteocytes and blood vessels to grow inward. Important factors that determine the successful regeneration of tissues and organs include surface chemistry, porosity, pore microstructure and macrostructure, and stent shape.

U.S. Patent No. 20030055512 provides an injectable and moldable bone cement comprising a biodegradable calcium-based compound comprising calcium sulfate, hydroxyapatite and tricalcium phosphate. However, this patent application does not provide a bioabsorbable stent.

WO 2005/105170 relates to bone replacement compositions and methods of use. In a preferred embodiment, the composition comprises anhydrous calcium sulfate, calcium sulfate dihydrate, and polyethylene glycol (PEG). CN 1724081 A provides a composite porous calcium sulphate scaffold having a polymer, wherein a polylactic acid or a lactic acid/alkyd copolymer or a polybasic acid or polycaprolactone or a polyhydroxybutyrate or a polyhydroxybutyrate is copolymerized The compound or polyanhydride is dissolved in chloroform, stirred, mixed with calcium sulfate in proportion, poured into a mold and dried to prepare the composite. US 2002018797 (A1) relates to a nano-calcium phosphate/collagen complex simulating natural bone in composition and microstructure, and from the complex with poly(lactic acid) (PLA) or poly(lactic acid co-glycolic acid) Porous bone substitutes and tissue engineering scaffolds made of a composite of (PLGA). US 2008281431 provides a ceramic material that repairs defects in the bones of a human or animal individual, comprising a porous ceramic scaffold having a bioabsorbable coating and a carrier comprising denatured demineralized bone. Such a ceramic may contain a material selected from the group consisting of hydroxyapatite, tricalcium phosphate, calcium phosphate, calcium carbonate, calcium sulfate, and combinations thereof. However, the stents of the prior art described above do not provide sufficient compressive stress resistance.

For optimal bone regenerative capacity, it is preferred to use a porous scaffold having a three-dimensional (3-D) structure as a bone conduction matrix. Due to its spongy structure, porous scaffolds are generally unable to withstand many physiological loads in the early stages of bone defect treatment. Under load, the porous scaffold tends to deform and lose its pore structure, and thus may be detrimental to certain clinical applications where it is under pressure but still requires space. For example, an interbody fusion cage, a manual replacement used in a spinal fusion procedure, should have sufficient mechanical stability to support and transfer the load in order to maintain the intervertebral foramen height.

Therefore, there is still a need in the clinic to develop bioabsorbable composites having appropriate mechanical properties at the beginning of transplantation. After the composite is embedded in the bone defect, the porous structure of the composite scaffold is formed in situ by in vivo degradation.

It is an object of the present invention to provide a method of preparing a composite comprising calcium sulfate and polylactic acid comprising the steps of:

(a) dehydrating calcium sulfate selected from calcium sulfate dihydrate, calcium alpha-hemihydrate or calcium sulfate beta-hemihydrate or a mixture thereof to obtain anhydrous calcium sulfate;

(b) melting polylactic acid at a high temperature, and mixing the molten polylactic acid with anhydrous calcium sulfate to form a calcium sulfate-polylactic acid composite; wherein the ratio of polylactic acid to calcium sulfate is between about 80% and 50% (w/w) Up to about 20%-50% (w/w).

Another object of the invention is to provide a composite prepared by the process of the invention. There is also provided a composite comprising calcium sulfate and polylactic acid selected from the group consisting of calcium sulfate dihydrate, calcium sulfate alpha-hemihydrate or calcium sulfate beta-hemihydrate or a mixture thereof, wherein the ratio of polylactic acid to calcium sulfate is about 80%- 50% (w/w) is in the range of about 20% to 50% (w/w).

Another object of the present invention is to provide a method of forming a porous scaffold in situ comprising embedding the composite of the present invention into a bone defect and degrading the complex in vivo to form a porous scaffold.

Another object is to provide a method of preparing a porous composite comprising calcium sulfate and polylactic acid, comprising the steps of:

(a) dehydrating calcium sulfate selected from calcium sulfate dihydrate, calcium alpha-hemihydrate or calcium beta-hemihydrate or a mixture thereof to obtain anhydrous calcium sulfate;

(b) melting the polylactic acid at a high temperature, and mixing the molten polylactic acid with anhydrous calcium sulfate to form a calcium-polylactic acid composite;

(c) subjecting the calcium-polylactic acid composite to particle washing to form a porous composite having interconnected pores; wherein the ratio of polylactic acid to calcium sulfate is between about 80% and 50% (w/w) to about 20% In the range of -50% (w/w), and the pore size of the interconnected pores is in the range of 100 to 500 μm.

Another object is to provide a porous composite prepared by the process of the present invention. There is also provided a porous composite comprising calcium sulfate and polylactic acid selected from the group consisting of calcium sulfate dihydrate, calcium sulfate alpha-hemihydrate or calcium sulfate beta-hemihydrate or a mixture thereof, wherein the ratio of polylactic acid to calcium sulfate is about 80%. -50% (w/w) is in the range of about 20% to 50% (w/w), and wherein the pores are interconnected and the pore size is in the range of 100 to 500 μm.

The present invention provides a method of preparing a calcium sulfate-polylactic acid composite. Porous composites with more desirable mechanical properties prepared from calcium sulfate and polylactic acid in a specific ratio range. For example, it shows a high drop in strength and a Young's modulus. Due to its good mechanical properties, the composite can be used as an in situ perforated disc implant.

As used herein, "macropore" refers to a void formed by a polymer wall within a polymeric stent.

"Interconnection" means a flow path connecting macroscopic pores to each other. The interconnects comprise perforated interconnects (channels), microporous interconnects (channels), and nanopores of all of the materials defined above.

The "yield strength" of a material is defined in engineering and materials science as the stress at which a material begins to plastically deform. Prior to the point of surrender, the material is elastically deformed and will return to its original state when the applied stress is removed. Once the surrender point is exceeded, some parts will be permanently and irreversibly deformed.

"compressive strength" means the ability of a material to withstand axially directed thrust.

"Young's modulus" refers to the material properties that describe the hardness of a material and is therefore one of the most important properties in engineering design. The Young's modulus is also known as the tensile modulus, which is a measure of the hardness of an isotropic elastic material. It is defined as the ratio of uniaxial stress to uniaxial strain within the stress range controlled by Hooke's Law. This ratio can be determined experimentally from the slope of the stress-strain curve established during the tensile test of the material sample.

"Biodegradable" means the action of a biologically active organism or a natural metabolisable product that can be broken down into easily metabolized products.

"Implant" and the like mean that a foreign substance is placed in a patient's body during medical treatment such as disease, injury or trauma.

The term "bone defect" refers to any area of bone defect, such as voids, fissures, notches or other discontinuities in the bone. For example, a bone defect can be caused by or caused by a disease or trauma such as a pathological, inflammatory or neoplastic disease.

Method for preparing a composite comprising calcium sulfate and polylactic acid and a composite prepared therefrom

In one aspect, the present invention provides a method of preparing a composite comprising calcium sulfate and polylactic acid, comprising the steps of: (a) selecting a calcium sulfate dihydrate, a calcium sulfate hemihydrate or a beta-hemihydrate Calcium sulfate or a mixture thereof is dehydrated to obtain anhydrous calcium sulfate; and (b) molten polylactic acid is melted at a high temperature, and molten polylactic acid is mixed with anhydrous calcium sulfate to form a calcium-polylactic acid complex; wherein polylactic acid and sulfuric acid are mixed The ratio of calcium is about 80%-50% (w/w) 20%-50% (w/w) range.

In step (a) of the process of the invention, the calcium sulphate is dehydrated to obtain anhydrous calcium sulphate. This dehydration step is used to reduce the adverse effects of water in the process of the invention. In the process of melting polylactic acid, the presence of water will destroy the intermolecular ester bond and cause the cleavage of polylactic acid. Therefore, the high water content in the process will cause degradation of the polylactic acid and reduce the strength of the composite. Different forms of calcium sulfate can be used to control the crystal form and size of anhydrous calcium sulfate. According to the present invention, the calcium sulfate may be calcium sulfate dihydrate, calcium alpha-hemihydrate or calcium sulfate beta-hemihydrate or a mixture thereof. The calcium sulfate is preferably β-calcium sulfate hemihydrate.

In the step (b) of the present invention, the polylactic acid is melted at a high temperature and then mixed with anhydrous calcium sulfate to form a calcium-polylactic acid complex, wherein the ratio of the polylactic acid to the calcium sulfate is about 80% to 50% ( w/w) is in the range of about 20%-50% (w/w). The ratio of polylactic acid to calcium sulfate is preferably from about 75% to 50% (w/w) to about 25% to 50% (w/w), about 75% to 55% (w/w) to about 25% - 45% (w/w), about 75%-60% (w/w) to about 25%-40% (w/w) or about 75%-65% (w/w) to about 25%-35% Within the range of (w/w). The ratio of polylactic acid to calcium sulfate is more preferably 70% to 30% (w/w).

The temperature of the molten polylactic acid is known in the art. The elevated temperature is preferably in the range of from about 120 °C to about 300 °C. The elevated temperature is preferably from about 150 ° C to about 300 ° C, from about 150 ° C to about 280 ° C, from about 150 ° C to about 250 ° C, from about 180 ° C to about 300 ° C, from about 180 ° C to about 280 ° C, from about 180 ° C to about 250 °C or in the range of about 200 ° C to about 250 ° C. The elevated temperature is more preferably in the range of from about 200 ° C to about 250 ° C.

In another aspect, the invention provides a method of preparing by the method of the invention Complex. The composite of the present invention comprises calcium sulfate and polylactic acid selected from calcium sulfate dihydrate, calcium sulfate alpha-hemihydrate or calcium sulfate beta-hemihydrate or a mixture thereof, wherein the ratio of polylactic acid to calcium sulfate is about 80%-50 The %(w/w) ratio is in the range of about 20% to 50% (w/w). The ratio of polylactic acid to calcium sulfate is preferably from about 75% to 50% (w/w) to about 25% to 50% (w/w), about 75% to 55% (w/w) to about 25% - 45% (w/w), about 75%-60% (w/w) to about 25%-40% (w/w) or about 75%-65% (w/w) to about 25%-35% Within the range of (w/w). The ratio of polylactic acid to calcium sulfate is more preferably 70% to 30% (w/w). According to the invention, the calcium sulfate is preferably beta-calcium sulfate hemihydrate.

The complex of the present invention has an initial mechanical property sufficient to maintain the stability of the bone defect area for a long period of time. The composite of the present invention is biodegradable and has a high drop in strength and a Young's modulus. Thus, the composite has suitable mechanical properties to allow for the formation of new bones. In addition, the composite has a higher degradation rate than polylactic acid and, therefore, provides more space for the growth of new bones and thereby improves the stability of bone fusion.

Method for forming a porous stent in situ

In another aspect, the invention also provides a method of forming a porous scaffold in situ comprising embedding a complex of the invention into a bone defect and decomposing the complex in vivo to form a porous scaffold. In one embodiment, the porous scaffold is a spinal vertebral cage. Transplantation of the complex of the invention is a standard surgical technique using bone repair or replacement. The complex can be directly transplanted to a site where bone growth is required. In a preferred embodiment, the complex is collapsed into a desired shape to repair a bone defect requiring treatment. The initial strength of the composite is sufficient to maintain the stress at the initial stage of the graft. After transplanting for a long time, due to polylactic acid and sulfuric acid The different degradation rates of calcium, the complex forms a porous configuration with a variety of pore sizes (including macroscopic pores, microscopic pores, and nanopores) and the pores are interconnected structures. Therefore, the purpose of the PLA/CS stent system for in situ pore formation is to provide mechanical stability during the early stages of healing. Thereafter, the calcium ions released from the dissolution of calcium sulfate and the resulting pore structure provide further advantageous conditions for osteocytes and blood vessels to grow inward.

Method for preparing porous composite comprising calcium sulfate and polylactic acid and porous composite prepared therefrom

In another aspect, the present invention provides a method of preparing a porous composite comprising calcium sulfate and polylactic acid, comprising the steps of: (a) selecting a calcium sulfate dihydrate, calcium sulfate alpha-hemihydrate or beta- Calcium sulfate hemihydrate or a mixture thereof is dehydrated to obtain anhydrous calcium sulfate; (b) molten polylactic acid is melted at a high temperature, and molten polylactic acid is mixed with anhydrous calcium sulfate to form a calcium-polylactic acid complex; and (c) The calcium-polylactic acid composite is subjected to particle washing to form a porous composite having interconnected pores; wherein the ratio of polylactic acid to calcium sulfate is from about 80% to 50% (w/w) to about 20% to 50% Within the range of (w/w), and the pore size of the interconnected pores is in the range of 100 to 500 μm.

Step (a) and step (b) and the examples thereof are consistent with those mentioned in the method of preparing a composite comprising calcium sulfate and polylactic acid, wherein the ratio of polylactic acid to calcium sulfate is about 80% - 50 The %(w/w) ratio is in the range of about 20% to 50% (w/w). The ratio of polylactic acid to calcium sulfate is preferably from about 75% to 50% (w/w) to about 25% to 50% (w/w), about 75% to 55% (w/w) to about 25% - 45% (w/w), approx. 75%-60% (w/w) is in the range of about 25% to 40% (w/w) or about 75% to 65% (w/w) to about 25% to 35% (w/w). The ratio of polylactic acid to calcium sulfate is more preferably 70% to 30% (w/w). With regard to step (c), the calcium-polylactic acid composite is subjected to particle washing to form a porous composite having interconnected pores. Particle elution techniques have been widely used to make 3D porous scaffolds for tissue engineering applications. Briefly, particle washing involves the manufacture of a suspension of polymeric composites in a solvent. The preferred solvent is water, most preferably distilled deionized water which does not dissolve the polymer or cause significant polymer hydrolysis during the time required for treatment. The porogen particles (such as salts, gelatin or waxy hydrocarbon particles) are ground and sieved into small particles and the particles of the desired size are transferred to the mold. The polymer suspension is then cast into a porogen filled mold. The solvent is then removed by evaporation under atmospheric pressure and/or vacuum. After evaporation of the solvent, the porogen crystals are filtered by immersion in water to form a porous structure.

According to the present invention, the pore size of the interconnected pores of the porous composite is in the range of 100 to 500 μm. The pore size is preferably in the range of 150 to 450 μm or 200 to 400 μm. In a preferred embodiment of the invention, sodium chloride (NaCl) is used as the porogen and the concentration of NaCl remaining in the particle wash of step (c) is less than 10 ppm.

In another aspect, the invention provides a porous composite prepared by the method of the invention. The porous composite of the present invention comprises calcium sulfate and polylactic acid selected from calcium sulfate dihydrate, calcium sulfate alpha-hemihydrate or calcium sulfate beta-hemihydrate or a mixture thereof, wherein the ratio of polylactic acid to calcium sulfate is about 80%- The 50% (w/w) ratio is in the range of about 20% to 50% (w/w), and wherein the pores are interconnected and the pore size is in the range of 100 to 500 μm. The ratio of polylactic acid to calcium sulfate Preferably, the ratio is about 75%-50% (w/w), about 25%-50% (w/w), about 75%-55% (w/w), about 25%-45% (w/w), From about 75% to 60% (w/w) to about 25% to 40% (w/w) or about 75% to 65% (w/w) to about 25% to 35% (w/w) . The ratio of polylactic acid to calcium sulfate is more preferably 70% to 30% (w/w). The pore size is preferably in the range of 150 to 450 μm or 200 to 400 μm. According to the invention, the calcium sulfate is preferably beta-calcium sulfate hemihydrate.

The composite may have a porous structure that reduces the initial ultimate compressive stress of the composite and increases the rate of degradation of the composite. Surprisingly, the porous composite of the present invention provides a higher rate of cell adhesion, and cells cultured in the complex have higher alkaline phosphatase activity and higher osteopontin (OPN) and osteophytes Bone sialoprotein (BSP) mRNA expression.

Instance Example 1 Preparation of the composite of the present invention

The polylactic acid and the calcium sulfate beta-hemihydrate are dehydrated by drying. The polylactic acid was placed in an oven and dried at a temperature of about 50 ° C for 2 days. The β-hemihydrate calcium sulfate was dried at a temperature of about 500 ° C for 1 hour, then transferred to an oven and dried at 50 ° C for 2 days. The dried β-hemihydrate calcium sulfate is added to the molten polylactic acid at a ratio of 20% to 80%, 30% to 70% or 40% to 60% by heating the melt-dried polylactic acid, and at about 220 ° C. The mixture is mixed at a temperature to obtain a composite of the present invention.

Example 2 Preparation of the Porous Composite of the Present Invention

The porous composite of the present invention was prepared using the composite of polylactic acid and β-hemihydrate calcium sulfate in a ratio of about 70% to about 30% obtained in Example 1. The composite of Example 1 was heated to about 220 ° C, followed by the addition of NaCl. The resulting porous composite was filled into a circular mold and then cooled to room temperature. The cooled porous composite was heated at 110 ° C for 4 hours, followed by cooling to room temperature. The resulting porous composite was taken out from the mold and the surface of the composite was rubbed with a sandpaper. Subsequently, the porous composite was immersed in distilled water to remove NaCl. The resulting porous composite was dried in an oven at a temperature of about 50 ° C for 1 day.

Example 3 Compressive Strength Test of the Composite of the Invention

The compressive strength was measured by ASTM D695 according to the compressive strength test standard by using a hydraulic material testing machine. A compressive strength test was performed on a composite prepared as described in Example 1 containing a ratio of 20% to 80%, 30% to 70% or 40% to 60% of calcium sulfate hemihydrate and polylactic acid. The compressive strength test was further carried out at 0, 1, and 3 months for a composite containing a ratio of 30% to 70% of calcium sulfate hemihydrate and polylactic acid. 1(A) and (B) show the compressive stress (A) of a composite of β-hemihydrate calcium sulfate and polylactic acid having a ratio of 20% to 80%, 30% to 70% or 40% to 60%. Young's modulus (B). The above composite exhibits favorable compressive stress. 2(A) and (B) show that after one month, there is no significant change in compressive stress (A) and Young's modulus (B), so the composite of the present invention can maintain stability for at least one month (P: polylactic acid) Composite; PC: Example 1 comprises a composite of a ratio of 30% to 70% of calcium sulfate hemihydrate and polylactic acid). After 3 months, the composite retained acceptable stress despite the degradation.

The porous composite prepared in Example 2 was subjected to a compressive strength test during 0 to 3 months. 3(A) and (B) show compressive stress (A) and Young's modulus (B) during 0 to 3 months (pP: porous polylactic acid; pPC: porous composite of Example 2). After 1 month, the compressive stress and Young's modulus of the porous composite of Example 2 did not change significantly, and thus the porous composite of the present invention can maintain stability for at least one month. After 3 months, the composite retained acceptable stress despite the degradation exhibited.

Example 4 Information on the expression of ribonucleic acid (mRNA) of osteopontin (OPN) and bone sialoprotein (BSP) in osteoblasts inoculated with the composite of the present invention and porous composites

Osteoblasts obtained from the skull of a 2 day old Sprague Dawley rat were inoculated and attached to the composite of Example 1 and the porous composite of Example 2. The cells were cultured for 3 weeks with α-MEM containing 50 μg/ml of L-ascorbic acid 2-phosphate, 10 mM β-glycerophosphate and 10% FBS. Use QIAGEN RNeasy according to the manufacturer's instructions Microcapsules (Cat. No. 74104; QIAGEN, CA, USA) were used to measure the amount of mRNA for OPN and BSP mRNA of cells in the complex of Example 1 and the porous complex of Example 2. 4 shows that the osteoblasts cultured in the complex of the present invention and the porous composite exhibit high expression levels of OPN mRNA and BSP mRNA (P: polylactic acid complex; pP: porous polylactic acid; PC: the inclusion ratio of Example 1 is 30% to 70% composite of β-hemihydrate calcium sulfate and polylactic acid; pPC: porous composite of Example 2; B: black; W1: week 1; W2: week 2; W3: week 3) .

Example 5 Method for forming a porous scaffold in situ

A 28.3 mm ² defect was drilled in the lower jaw of the Lanyu pig. A scaffold of the polylactic acid complex scaffold and the scaffold of the β-hemihydrate calcium sulfate and polylactic acid of Example 1 were separately implanted into the defect. After 8 weeks, as can be seen from the results of the x-ray photograph (see Figure 5), the size of the defect implanted in the polylactic acid stent was reduced to 16.3 ± 2.0 mm ² , and the size of the defect implanted in the polylactic acid and calcium sulfate stent was reduced. As small as 8.3 ± 0.9 mm ² . Clearly, the composite of Example 1 produced an unexpected effect relative to the polylactic acid stent.

1(A) and (B) show the compressive stress (A) of a composite of β-hemihydrate calcium sulfate and polylactic acid having a ratio of 20% to 80%, 30% to 70% or 40% to 60%. Young's modulus (B);

2(A) and (B) show compressive stress (A) and Young's modulus (B) of a composite of β-hemihydrate calcium sulfate and polylactic acid having a ratio of 30% to 70% during 3 months. ;

Figure 3 (A) and (B) show the compressive stress (A) and Young's modulus (B) during 0 to 3 months;

Figure 4 shows that the osteoblasts cultured in the composite of the present invention and the porous composite exhibit high expression levels of OPN mRNA and BSP mRNA;

Figure 5 shows the reduction in size of the defect implanted with the polylactic acid and calcium sulfate scaffold.

Claims (12)

A method of preparing a porous composite comprising calcium sulfate and polylactic acid, comprising the steps of: (a) setting a calcium sulfate selected from the group consisting of calcium sulfate dihydrate, calcium sulfate alpha-hemihydrate or calcium sulfate beta-hemihydrate or a mixture thereof Dehydrating to obtain anhydrous calcium sulfate; (b) melting polylactic acid at a high temperature, and mixing the molten polylactic acid with anhydrous calcium sulfate to form a calcium-polylactic acid complex; and (c) performing the calcium-polylactic acid composite The particles are washed to form a porous composite having interconnected pores; wherein the ratio of polylactic acid to calcium sulfate is in the range of from about 80% to about 50% (w/w) to about 20% to 50% (w/w), And the pore size of the interconnected pores is in the range of 100 to 500 μm. The method of claim 1, wherein the calcium sulfate in the step (a) is β-calcium sulfate hemihydrate. The method of claim 1, wherein the ratio of polylactic acid to calcium sulfate is from about 75% to 50% (w/w) to about 25% to 50% (w/w), about 75% to 55% (w/w). a ratio of about 25% to 40% (w/w), about 75% to 60% (w/w) to about 25% to 45% (w/w) or about 75% to 65% (w/w) It is in the range of about 25% to 35% (w/w). The method of claim 1, wherein the ratio of polylactic acid to calcium sulfate is 70% to 30% (w/w). The method of claim 1, wherein the elevated temperature in step (b) is in the range of from about 120 °C to about 300 °C. The method of claim 1, wherein the high temperature in step (b) is from about 150 ° C to about 300 ° C, from about 150 ° C to about 280 ° C, from about 150 ° C to about 250 ° C, from about 180 ° C to about 300 ° C, about From 180 ° C to about 280 ° C, from about 180 ° C to about 250 ° C or from about 200 ° C to about 250 ° C. The method of claim 1, wherein the elevated temperature in step (b) is in the range of from about 200 °C to about 250 °C. A porous composite comprising calcium sulfate and polylactic acid selected from the group consisting of calcium sulfate dihydrate, calcium sulfate alpha-hemihydrate or calcium sulfate beta-hemihydrate or a mixture thereof, wherein the ratio of polylactic acid to calcium sulfate is about 80%-50 The % (w/w) ratio is in the range of about 20% to 50% (w/w), and wherein the pores are interconnected and the pores have a size in the range of 100 to 500 μm. The porous composite of claim 8, wherein the ratio of polylactic acid to calcium sulfate is in the range of from about 75% to 50% (w/w) to about 25% to 50% (w/w). The porous composite of claim 8, wherein the ratio of polylactic acid to calcium sulfate is 70% to 30% (w/w). The porous composite of claim 8, wherein the calcium sulfate is beta-hemihydrate calcium sulfate. The porous composite of claim 8, wherein the pore size is in the range of 150 to 450 μm or 200 to 400 μm.