TWI438276B - Poly (lactic-glycolic) acid cross linked alendronate (plga-aln) a short term controlled release system for stem cell differentiation and drug delivery - Google Patents

Description

Polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) short-term controlled release composition for guiding stem cell differentiation and drug delivery

This invention relates to a short-term controlled release composition and a method of making the same. More specifically, the present invention relates to a composition that can be applied to the technical field of stem cell tissue engineering.

Inducing factors play a major role in inducing stem cell differentiation into tissue-specific cells, so they can be applied to tissue engineering (Lutolf and Hubbell, Nat Biotechnol, 2005, 23: 47-55). The inducing factor can be a protein or a chemical (Gaissmaier et al, Injury, 2008, Suppl 1: S88-96; Zur Nieden et al, BMC Dev Biol, 2005, 5:1); however, these inducing factors have their disadvantages. Where it is expensive, it can damage tissue or be difficult to transport. Therefore, it is important to find an inducing factor that can initiate and/or promote stem cell differentiation to cause subsequent deposition of a particular matrix, which can cause regeneration in vivo. It has been reported that bone morphogenetic protein-2 (BMP-2) plays an important role in the early differentiation of adult stem cells into osteoblasts or chondrocytes (Chen et al, Growth Factors, 2004, 22). :233-241; Shea et al, J Cell Biochem, 2003, 90: 1112-1127; Kato et al, Life Sci, 2009, 84: 302-310). Previous reports also indicated that bone morphogenetic protein-2 (BMP-2) induces differentiation of mesenchymal stem cells (MSCs) and promotes in vivo and in vitro repair of bone and cartilage (Gaissmaier et al, Injury, 2008, Suppl 1: S88-96; Zhao et al, J Control Release, 2010, 141: 30-37; Diekman et al, Tissue Eng Part A, 2009; Mrugala et al, Cloning Stem Cells, 2009, 11: 61-76; Park et al, J Biosci Bioeng, 2009, 108: 530-537 Hou et al, Biotechnol Lett, 2009, 31: 1183-1189).

Bisphosphonates are commonly used in the treatment of osteoporosis (Russell, Pediatrics, 2007, 119 Suppl 2: S150-162; Rogers, Curr Pharm, 2003, 9: 2643-2658; Fisher et al, Endocrinology, 2000) , 141: 4793-4796). Alendronate (ALN) is one of the bisphosphonates that act by interfering with the mevalonate pathway in osteoblasts. Recent reports have also indicated that alendronate stimulates mesenchymal stem cells (MSCs) to differentiate into a hard cell lineage. We investigated the effects of short-term treatment of human bone marrow mesenchymal stem cells (BMSCs) and adipose derived stem cells (ADSCs) with bisphosphonates. The results indicate that this treatment will be time-dependent. The time dependent manner improves the performance of intracellular bone morphogenetic protein-2 (BMP-2). We have also found that bisphosphonates enhance the differentiation of mesenchymal stem cells (MSCs) caused by the microenvironment into different lineages. We applied alanine phosphate (ALN) to adipose-derived stem cells (ADSCs) implanted in rat calvarial defects for a short period of time. The results showed that there was better hard bone repair after treatment with alendronate (ALN). We have also found that alendronate (ALN) enhances chondrogenesis induced by microenvironmental induction of human adipose stem cells (ADSCs) cultured in hyaluronic acid (HA) coating medium. However, a short-term sustained release carrier of alendronate (ALN) useful for stem cell tissue engineering is still controversial (Cartmell, J Pharm Sci, 2009, 98: 430-441).

Natural or synthetic polymeric carriers (microspheres or microspheres) have evolved into effective methods for controlling drug release (Cartmell, J Pharm Sci, 2009, 98: 430-441; Bhardwaj et al, J Diabetes Sci Technol, 2008, 2: 1016-1029; Mundargi et al, J Control Release, 2008, 125: 193-209). Excellent biocompatibility and biodegradability make poly(lactic-co-glycolic acid, PLGA) and poly(lactic acid) (PLA) It becomes a carrier more suitable for drug delivery (Lim et al, J Mater Sci Mater Med, 2009, 20: 1669-1675). Recently, our research report indicated that hyaluronic acid (HA) scaffold modified with polylactic acid-polyglycolic acid copolymer (PLGA) showed better chondrocyte differentiation-inducing effect on human adipose-derived stem cells ( h ADSCs) (Wu et al , Biomaterials, 2010, 31:631-640). Therefore, we hypothesized that polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) may be a better carrier for short-term sustained-release alendronate (ALN) and has enhanced human adipose stem cells ( h ADSCs) differentiation. potential.

One embodiment of the present invention provides a short-term controlled release composition comprising poly(lactic-glycolic acid cross linked alendronate, PLGA-ALN), wherein polylactic acid- Polyglycolic acid copolymer cross-linked alendronate is constructed to form a polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) or polylactic acid-polyglycolic acid copolymer cross-linked Lithium phosphate microspheres (PLGA-ALN-M).

The polylactic acid-polyglycolic acid copolymer crosslinked alendronate three-dimensional scaffold (PLGA-ALN-3D) of the short-term controlled release composition has a pore size of 150 to 300 μm and an average of 85% porosity.

The polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALN-M) of the short-term controlled release composition have a diameter of 50-100 μm and have a smooth surface.

The invention further provides a method of preparing the short-term controlled release composition comprising activating a carboxylic acid end group of a polylactic acid-polyglycolic acid copolymer (PLGA) to produce a carbodiimide hydrochloride/N-hydroxyamber Poly(lactic acid-polyglycolic acid copolymer (PLGA) activated by ethyl(dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide, EDC/NHS; and activated polylactic acid-polyglycolic acid copolymer (PLGA) Cross-linking reaction with sodium alendronate.

The method for preparing a short-term controlled release composition, wherein the carboxylic acid end group of the polylactic acid-polyglycolic acid copolymer (PLGA) is carbodiimide hydrochloride/ethyl-dimethylaminopropyl Carbodiimide/N-Hydroxysuccinimide, EDC/NHS) method activation.

The method of preparing a short-term controlled release composition, wherein the carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) process further comprises mixing N-hydroxysuccinimide (NHS), carbodiimide Hydrochloride (EDC) and polylactic acid-polyglycolic acid copolymer (PLGA). N-hydroxysuccinimide (NHS) and carbodiimide hydrochloride (EDC) were mixed at a ratio of 3 to 2; polylactic acid-polyglycolic acid copolymer (PLGA) was dissolved in dichloromethane. The polylactic acid-polyglycolic acid copolymer (PLGA) in which the carboxylic acid end group is activated in the carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) process is precipitated by excess diethyl ether.

A method of preparing a short-term controlled release composition, wherein the carbodiimide hydrochloride/N-hydroxyamber

Thiocarbamate (EDC/NHS) activated polylactic acid-polyglycolic acid copolymer (PLGA) and sodium alendronate are reacted together with a molar ratio. The crosslinking reaction is carried out in dry dimethysulphoxide.

Another embodiment of the present invention also provides a method of enhancing stem cell differentiation into a hard bone cell lineage comprising culturing stem cells in a microenvironment having a polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN).

The method for differentiating stem cells into a hard bone cell lineage, wherein a polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) is constructed to form a polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) or polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M). The polylactic acid-polyglycolic acid copolymer crosslinked alendronate three-dimensional scaffold (PLGA-ALN-3D) has a pore size of 150-300 micrometers and an average of 85% porosity. The polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALN-M) have a diameter of 50-100 μm and have a smooth surface.

In the method of fortifying stem cells to differentiate into an osteogenic lineage, stem cells refer to human adipose stem cells ( h ADSCs).

The invention also provides a method for enhancing stem cell differentiation into a chondrogenic lineage, comprising culturing stem cells with hyaluronan (HA) and polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) ) in the micro-environment.

The method for differentiating stem cells into chondrogenic lineage, wherein polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) is constructed to form a polylactic acid-polyglycolic acid copolymer crosslinked Lithium phosphate microspheres (PLGA-ALN-M). The polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALN-M) have a diameter of 50-100 μm and have a smooth surface. The method of fortifying stem cells to differentiate into a chondrogenic lineage, wherein the stem cells are human adipose stem cells ( h ADSCs).

Polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) enhances the hard bone and cartilage differentiation of induced human adipose-derived stem cells ( h ADSCs) in the presence of hard bone and cartilage, respectively. Moreover, the crosslinking between polylactic acid-polyglycolic acid copolymer (PLGA) and alendronate (ALN) does not affect the efficiency of alendronate (ALN). Therefore, polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) is a short-term controlled release vector that can enhance the differentiation of hard bone cells and chondrocytes of specific functional human adipose stem cells (committed h ADSC). For stem cell tissue engineering.

Example 1 Isolation and culture of human adipose-derived stem cells ( h ADSCs)

After obtaining the consent of all informed patients and the approval of the Kaohsiung Medical university hospital ethics committee, residual subcutaneous adipose tissue was obtained from the patient undergoing surgery. Human adipose-derived stem cells ( h ADSCs) were isolated from human subcutaneous adipose tissue by conventional methods (Fehrer and Lepperdinger, Exp Gerontol, 2005, 40: 926-930). The isolated human adipose-derived stem cells ( h ADSCs) were cultured in a K-NAC medium at 37 ° C, 5% CO ₂ and proliferated in a large amount, and the medium contained Keratinocyte-SFM (Gibco BRL, Rockville, MD) and supplemented with EGF-BPE. (Gibco BRL, Rockville, MD), N -Ethyl-L-cysteine, L-ascorbic acid-2-phosphate magnesium (Sigma, St. Louis, MO) and 5% FBS (Fehrer and Lepperdinger, Exp Gerontol, 2005, 40: 926-930).

Example 2 Synthetic polylactic acid-polyglycolic acid copolymer crosslinked alendronate (PLGA-linked)

The polylactic acid-polyglycolic acid copolymer crosslinked alendronate (PLGA-ALN) forming process comprises a two-step process; the first step is carbodiimide hydrochloride/N-hydroxysuccinimide The (EDC/NHS) method activates the carboxylic acid end group of the polylactic acid-polyglycolic acid copolymer (PLGA), and the second step is a crosslinking reaction. Briefly, 1 gram of polylactic acid-polyglycolic acid copolymer (PLGA, 50/50) was dissolved in 10 ml of dichloromethane and with a 3:2 ratio of carbodiimide hydrochloride (NHS). And a mixture of N-hydroxysuccinimide (EDC), and the reaction was stirred at room temperature for 2 hours. Next, the insoluble dicyclohexylurea was removed with a 0.45 mm pore size Teflon filter. After precipitating the activated polylactic acid-polyglycolic acid copolymer (PLGA) polymer product with an excess of diethyl ether, it was dried in vacuum for 4 hours. To prepare a polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN), activate the polylactic acid with a molar ratio of carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) - Polyglycolic acid copolymer (PLGA) and sodium alendronate were reacted together in dry dimethyl hydrazine and stirred at room temperature for 12 hours. The final product was precipitated and separated by addition of excess cold diethyl ether, separated and washed with distilled water. The obtained polylactic acid-polyglycolic acid copolymer crosslinked alendronate (PLGA-ALN) was dried in vacuo and stored at -20 °C.

Example 3 Forming Process and Characteristic Analysis of Porous Polylactic Acid-Polyglycolic Acid Copolymer Crosslinked Alendronate Three-dimensional Scaffold (PLGA-ALN-3D scaffolds)

Polylactic acid-polyglycolic acid copolymer crosslinked alendronate (PLGA-ALN) porous scaffold is prepared by salting out. Briefly, a 1:6 polylactic acid-polyglycolic acid copolymer crosslinked alendronate (PLGA-ALN) is mixed with a sodium chloride salt (particle size 300-400 microns) for magnetic stirring. Dissolved in 10 ml of chloroform. The colloidal precipitate was thoroughly mixed with the sieved salt particles, and placed in a dish-shaped Teflon mold having a thickness of 2 mm and a diameter of 5 mm, followed by partial evaporation of chloroform at room temperature to obtain a semi-solid object. The mold was successively immersed in a room temperature distilled aqueous solution, and the salt in the polymer/salt base was precipitated. The porous polymer scaffold was then taken out from the mold, washed three times with distilled water and dried in vacuum for 1 day. The overall morphology of the three-dimensional scaffold was observed by scanning electron microscopy (SEM) (Hitachi S3200, Tokyo, Japan); the polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) has 150-300 micron pore size and an average 85% porosity (Figures a, b).

Example 4 Preparation and Characterization of Polylactic Acid-Polyglycolic Acid Copolymer Crosslinked Alen Phosphate Microspheres (PLGA-ALN-M)

The polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALN-M) were produced by an oil emulsion technique (o/w emulsion technique). Briefly, a 10% polylactic acid-polyglycolic acid copolymer crosslinked alendronate (PLGA-ALN) polymer solution was obtained by dissolving in methylene chloride. The polymer solution was gradually added to 20 liters of a 1% polyvinyl alcohol liquid solution while vigorously stirring to prepare a single layer emulsion (o/w). The solution was stirred at room temperature for 30 minutes to harden the microspheres, followed by evaporation of the dichloromethane using a water pump, and the solid microspheres were collected by centrifugation. The resulting microspheres were washed three times with distilled water and lyophilized. The overall morphology of the microspheres was observed by scanning electron microscopy (SEM) (Hitachi S3200, Tokyo, Japan), and the average size of the microspheres was measured by a particle size analyzer; polylactic acid-polyglycolic acid copolymer crosslinked The bisphosphonate microspheres (PLGA-ALN-M) are 50-100 microns in diameter and have a smooth surface (Figures c, d).

Example 5 Release kinetics

10 mg of polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) or polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffolds (PLGA-ALN-3D The cells were suspended in 1 ml of phosphate buffered saline (PBS), and 500 μl of the solution was taken out from the mixture and replaced with fresh phosphate buffer (PBS). The alendronate (ALN) release concentration is measured by conventional spectrometry. The results of release kinetics experiments showed that polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) and polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA- ALN-M) maintains an effective concentration from 5 X 10 ^-7 moles to 5 X 10 ^-8 moles in 9 days (average daily concentration is 1 X 10 ^-7 moles) (Figure 3).

Example 6 Scanning electron microscopy (SEM) analysis

The morphological characteristics of the polylactic acid-polyglycolic acid copolymer crosslinked alendronate scaffolds (PLGA-ALN scaffolds) were observed by scanning electron microscopy (SEM) (SEM, JEOL, Tokyo, Japan). However, the sample must first be coated with a layer of gold at the ambient temperature with the nozzle applicator. Micrographs of both stents were taken at 50x and 100x magnification. The overall morphology of the stent was observed after the stent sample was coated with gold. The scaffold (PLGA-ALN-3D) has an average pore size of 150-300 microns and an average porosity of 85% (Figures a, b). The polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALN-M) have a smooth surface with a diameter of 50-100 μm (Figure 1 c, d).

Example 7 Cultured cells on a polylactic acid-polyglycolic acid copolymer three-dimensional scaffold (PLGA-3D) and a polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D)

Preparation of polylactic acid-polyglycolic acid copolymer three-dimensional scaffold (PLGA-3D) and polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) and human adipose stem cells ( h ADSCs) Cell/stent construct. Pre-wet polylactic acid-polyglycolic acid copolymer three-dimensional scaffold (PLGA-3D) and polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) to 70% (v/v) The ethanol liquid solution was sterilized (Yoon et al, Biotechnol Bioeng, 2002, 78: 1-10; Yoon et al, Biomaterials, 2004, 25: 5613-5620) and placed in a 24-well dish. 100 microliters of the cell suspension (3x10 ⁵ cells / [mu] l) of each uploaded to the stent pre-wetted and penetrated into the scaffold until the cells. The cell/scaffold construct was then incubated for 4 hours at 37 ° C under 5% CO ₂ for cell attachment. After cell attachment, the cell/stent construct was exchanged into another new 24-well plate to remove residual cells from the bottom and 1 ml was added to each cell/stent construct that was replaced with a new 24-well plate. Medium. Standard medium: basal medium (DMEM) containing 10% FBS (Hyclone, Logan, UT), 1% non-essential amino acid, and 100 units/ml penicillin/streptomycin (Gibco-BRL, Grand Island, NY); The medium needs to be replaced every other day and it must be shaken continuously during the cultivation. Cell/scaffold constructs were collected at each indicated time point for further experiments.

Example 8 Attachment and survival test of polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) and polylactic acid-polyglycolic acid copolymer three-dimensional scaffold (PLGA-3D)

To test cell adhesion, cells were attached to a polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) or polylactic acid-polyglycolic acid (PLGA) scaffold for 4 hours, then rinsed for cell/stent construction Remove and remove it from the 24-well plate. To obtain the number of viable cells attached to each scaffold within the first 4 hours, the number of viable cells not attached to the wells was counted and compared to the control group (24-well plates inoculated with cells but without scaffolds). Use CellTiter AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI), a colorimetric method for determining the number of viable cells in a medium, was used to calculate the number of cells (Relic et al, J Immunol, 2001, 166: 2775-2782). Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) was cultured in human wells ( h ADSCs) Transformation of mitochondrial activity into a (Formazan) (Relic et al, J Immunol, 2001, 166: 2775-2782; Ma et al, Biomaterials, 2007, 28: 1620-1628; Magne et al, J Bone Miner Res, 2003, 18: 1430-1442) And released into the medium The amount of (formazan) product is directly proportional to the number of viable cells in the medium, and the amount of the product can be determined by the light absorption value at a wavelength of 490 nm (Relicetal, J Immunol, 2001, 166: 2775-2782). At the specified time point, the freshly prepared MTS reaction mixture was diluted in a standard medium at a ratio of 1:5 (MTS: medium), and the diluted reaction mixture was added to the wells in which the cells were cultured, and Additional incubation was continued for 4 hours at 37 ° C, 5% CO ₂ . After 4 hours of additional incubation, 100 μl of medium containing the transformed MTS was removed from each well and placed in a 96-well plate using a microplate reader (PathTech) and KC junior software. The absorbance at 490 nm wavelength was recorded. The cell attachment rate of human adipose-derived stem cells ( h ADSCs) is calculated by the following formula:

Cell attachment rate (%) = [1 - (number of cells not attached to the stent / number of cells in the control well)] x 100%

The results showed that 80% of human adipose-derived stem cells (hADSCs) were attached to a polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D), and the results were significantly correlated with polylactic acid-polygans The alkyd copolymer three-dimensional scaffold (PLGA-3D) is similar (figure one e).

To test cell viability, after attaching the cells to the scaffold, switch the cell/stent construct to another new plate and continue to incubate 1 or 3 in standard medium at 37 ° C, 5% CO ₂ day. At each given time point, the freshly prepared MTS reaction mixture was diluted in standard medium at a ratio of 1:5 (MTS: medium), and the diluted reaction mixture was added to the wells in which the cells were cultured. And calculate the number of living cells in the construct. The results of the cell killing test (MTS assay) showed that whether the polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) or the polylactic acid-polyglycolic acid copolymer crosslinked Human three-dimensional scaffolds (PLGA-ALN-3D) treated human adipose-derived stem cells ( h ADSCs) with no deleterious toxic effects on day 1 or 3 (Figure 2).

Example 9 Osteogenic differentiation

Human adipose-derived stem cells ( h ADSCs) were seeded on a polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) at a cell density of 10 ⁵ cells/well. After 12 hours of incubation, conditioning medium was added. (conditioned medium) (10% FBS, 100 units/ml penicillin and 100 g/ml streptomycin were added to DMEM) and cultured in an incubator at 37 ° C, 5% CO 2 for 7 days. After 7 days, the medium was changed to osteoinduction medium and replaced every 2 to 3 days; after 14 days, cells were fixed with 4% paraformaldehyde and tested for osteosynthesis by erythromycin S staining. . The results of rutin S staining showed that human adipose-derived stem cells ( h ADSCs) treated with polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) were cultured on days 7 and 14 The degree of mineralization was higher than that of the untreated control cells (Figure 4).

Example 10 Alizarin red S staining

Alizarin red S staining was used to determine the extent of extracellular matrix (ECM) calcification after three weeks of hard bone differentiation induction. The cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. After washing once with distilled water, 1 ml of a solution of ruthenium S (1% in distilled water, pH 4.2) was added to the well of each 12-well plate. The dye solution was removed after 10 minutes and each well was washed 4 to 5 times with clean water (H ₂ O). The plate that was fixed and dyed was then air dried at room temperature. To quantify the degree of mineralization by spectrophotometry (wavelength: 415 nm), cell-bound alizarin red S is dissolved in 10% acetic acid (Figure 4).

Example 11 Osteoblast differentiation of human adipose-derived stem cells ( h ADSCs)

To assess the osteoblast differentiation of human adipose-derived stem cells ( h ADSCs), real-time PCR was used to detect the expression of osteogenic marker genes (RNA) in cells cultured on scaffolds. The mRNAs of the hard bone marker genes (osteocalcin, bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), and Runt-related transcription factor (Runx2) are expressed in the treatment of polylactic acid-polyglycolic acid. Copolymer cross-linked alendronate microspheres (PLGA-ALN-M), 1, 3, and 5 days of human adipose-derived stem cells ( h ADSCs) were significantly increased compared with the control group (p>0.05). Show five).

Example 12 Chondrocyte differentiation of human adipose-derived stem cells ( h ADSCs)

The human adipose-derived stem cells ( h ADSCs) were cultured in a pre-coated hyaluronic acid migration chamber (HA pre-coated trans-well fitted 24 well plate), and the inoculation culture density was 10 ⁵ cells/well, followed by culture 2 Hours to form three-dimensional high-density micromass; 20 μl of polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) (100) through a migration chamber (migration chamber) Mg/ml), adding conditioning medium (DMEM/10% FBS, 50 nM ascorbic acid-2 phosphate, 1% antibiotic/antimycotic), and incubating in an incubator at 37 ° C, 5% CO 2 for 14 days. After 7 days, the trans-well containing the polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) was removed and replaced every 2 to 3 days. Medium (Figure 6).

Example 13 RNA isolation and quantitative polymerase chain reaction (Real-time polymerase chain reaction, real-time PCR)

Cells were harvested from the cell/scaffold construct at the indicated time points. The RNA of the collected cells was extracted using TRIzol (Gibco BRL, Rockville, MD) according to the product operation manual. Briefly, 0.5-1 micrograms of total RNA was reverse transcribed into complementary DNA (cDNA) by SuperScript First-Strand Synthesis System (Invitrogen) per 20 microliters of reaction volume. Perform quantitative real-time PCR reactions and use iQ ^TM SYBR Supermix (Bio-Rad Laboratories Inc, Hercules, CA) was monitored and quantified using a real-time PCR detection system (Bio-Rad Laboratories Inc, Hercules, CA). Analysis of complementary gene (cDNA) samples (2 microliters of cDNA sample in a total volume of 25 μl of each reaction) and reference gene, glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3) -phosphate-dehydrogenase, GAPDH). The performance of each target gene was calculated in a conventional manner using 2- ^ΔΔCt (Livak and Schmittgen, Methods, 2001, 25(4): 402-408). Four readings of each target gene were obtained from each experimental sample, and the experiments were repeated at least three times. The mRNAs of osteoclast marker genes (osteocalcin, bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), and Runt-related transcription factor (Runx2) are expressed in the treatment of polylactic acid-polyglycol The acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M), 1, 3 and 5 days of human adipose-derived stem cells ( h ADSCs) showed a significant increase (p>0.05) compared with the control group. Figure 5). Chondrocyte marker genes, such as bone morphogenetic protein-2 (BMP-2), SOX-9, collagen type II (collagen type II) and proteoglycan (Aggrecan), under hyaluronic acid microenvironment Treatment of polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) 1, 3 and 5 days of human adipose-derived stem cells ( h ADSCs) were significantly higher than those of the control group. The rise (p>0.05) (Figure 7).

Example 14 Animals and surgery

All animal experiments were performed in accordance with the Kaohsiung Medical University Animal Care and Use Committee guidelines (IRB). Eighteen male Sprague Dawley rats (250-300 g) weighing 8 to 10 weeks of age were housed in a light and temperature controlled environment and given food and water. Rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. The hair on the dorsal side of the skull was removed for pre-operative aseptic preparation and an approximately 20 mm, anterior incision (sagittal incision) was opened on the animal's scalp. The periosteum was removed and a 5 mm diameter skull defect was made with a non-flushing slow drill, causing thermal damage to the host bone edge without damaging the dura mater. Skeletal defects were randomly implanted in a polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) or a polylactic acid-polyglycolic acid copolymer three-dimensional scaffold (PLGA-3D) Human adipose-derived stem cells ( h ADSCs) or left vacancies (n=6). After the incision was sutured, the animals were allowed to recover for 8 weeks after surgery and sacrificed by inhalation of CO ₂ after recovery. To collect the implant, open the skin and remove the defect from the surrounding bone. After the sample is fixed, it is ready for micro-focus computer tomography analysis (hCT analysis) and histology analysis. Radiographic display showed that the polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) construct had better bone growth after 8 weeks of implantation in the rat skull defect (Figure 8). . Observations by micro-focus computed tomography (μCT analysis) showed that the rat skull defect was treated with a polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) construct. Bone formation has a good effect (Figure 9).

Example 15 Statistical analysis

Three independent culture samples were tested for each biochemical analysis. Each experiment was repeated at least three times and the results of representative experiments (expressed as mean ± SEM) were presented. Statistical significance was assessed using one-way analysis of variance (ANOVA) and multiple comparative analyses were performed using Scheffe's method. It was considered to be significant when p < 0.05.

The above embodiments are merely illustrative of the principles of the invention and its advantages, and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be as listed in the scope of the application specific examples described later.

Figure 1 is a (a) polylactic acid-polyglycolic acid copolymer scaffold (PLGA scaffolds), (b) polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D), (c Scanning electron microscopy of polylactic acid-polyglycolic acid copolymer microspheres (PLGA microspheres) and (d) polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALN-M) SEM) Image (e) cell patching ratio of polylactic acid-polyglycolic acid copolymer scaffold (PLGA) and polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D).

Figure 2 is a quantitative result of analyzing cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS).

Figure 3 is a graph showing the release profile of alendronate (ALN) from a polylactic acid-polyglycolic acid copolymer crosslinked alendronate (PLGA-ALN) carrier.

Figure 4 shows the determination of human adipose-derived stem cells ( h ADSCs) treated with polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) by Alizarin red S staining. The staining situation (A) and its quantitative result (B).

Figure 5 shows the treatment of human adipose-derived cells ( h ADSCs) of polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) by reverse transcription polymerase chain reaction (RT-PCR analysis). The situation of the osteogenic gene: (a) bone morphogenetic protein-2 (BMP-2); (b) Runt-related transcription factor (Runx2); (c) alkaline phosphatase (ALP) and d) Osteocalcin.

Figure 6 shows human fat stem cells (hADSCs) cultured in hyaluronic acid (HA) microenvironment, treated with polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M) for 2 hours. Chondrogenesis is enhanced by cell aggregation.

Figure 7 is a diagram showing the expression of chondrogenic genes by human adipose-derived stem cells ( h ADSCs) cross-linked with a polylactic acid-polyglycolic acid copolymer (PLGA-ALN-M): (a) bone Morphogenetic protein-2 (BMP-2); (b) SOX-9; (c) collagen type II (collagen type II) and (d) proteoglycan (Aggrecan).

Figure 8 presents the use of (a) polylactic acid-polyglycolic acid copolymer (PLGA), and (b) polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) Human adipose-derived stem cells (hADSCs), radiographic images after eight weeks of treatment of rat calvarial defects.

Figure 9 presents the use of (a) polylactic acid-polyglycolic acid copolymer (PLGA), and (b) polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) Human fat stem cells (hADSCs) were analyzed by micro computed tomography (μCT) after eight weeks of treatment of rat calvarial defects.

While the invention has been described in detail and illustrated by the embodiments of the invention

It will be readily apparent to those skilled in the art that the present invention is capable of <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The processes and methods for generating them are representative of the preferred embodiments, are illustrative, and are not intended to limit the scope of the invention. Modifications and other uses are readily apparent to those skilled in the art. These modifications are also encompassed within the spirit of the invention and are defined by the scope of the claims.

Claims (18)

A short-term controlled release composition that maintains an effective concentration of 5 x 10 ^-7 to 5 x 10 ^-8 moles of Alendronate for at least 9 days, comprising a polylactic acid-polyglycolic acid copolymer cross-linked alendronate (poly(lactic-co-glycolic acid) cross-linked alendronate, PLGA-ALN), which is constructed as a polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGAALN-3D) or polylactic acid-poly Glycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M), the polylactic acid-polyglycolic acid copolymer cross-linked alendronate three-dimensional scaffold (PLGA-ALN-3D) has a pore size of 150-300 microns And an average of 85% porosity; the polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALNM) have a diameter of 50-100 microns. A method for preparing a short-term controlled release composition of claim 1, comprising activating a carboxylic acid end group of a polylactic acid-polyglycolic acid copolymer (PLGA) to produce a carbodiimide hydrochloride/N-hydroxyl group Poly(lactic acid-polyglycolic acid copolymer (PLGA) activated by ethyl(dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide, EDC/NHS; and activated polylactic acid-polyglycolic acid copolymer (PLGA) Cross-linking reaction with sodium alendronate. The method of claim 2, wherein the carboxylic acid end group of the polylactic acid-polyglycolic acid copolymer (PLGA) is carbodiimide hydrochloride/N-hydroxysuccinimide (ethyl (dimethylaminopropyl) carbodiimide / N-Hydroxysuccinimide, EDC / NHS) method activation. The method of claim 3, wherein the carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) method further comprises mixing N-hydroxysuccinimide (NHS), carbon two Imine hydrochloride (EDC) and polylactic acid-polyglycolic acid copolymer (PLGA). The method of claim 4, wherein N-hydroxysuccinimide (NHS) and carbodiimide hydrochloride (EDC) are mixed in a ratio of 3 to 2. The method of claim 4, wherein the polylactic acid-polyglycolic acid copolymer (PLGA) is dissolved in dichloromethane. The method of claim 3, wherein the polylactic acid-polyglycolic acid copolymer (PLGA) in which the carboxylic acid end group is activated is precipitated by excess diethyl ether. The method of claim 2, wherein the carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS)-activated polylactic acid-polyglycolic acid copolymer (PLGA) and alendronate The sodium alendronate is reacted together with the molar ratio. The method of claim 2, wherein the crosslinking reaction is carried out in dry dimethysulphoxide. A method for enhancing differentiation of stem cells into a hard bone cell lineage comprises culturing stem cells in a microenvironment of a polylactic acid-polyglycolic acid copolymer cross-linked alendronate (PLGA-ALN) having the first aspect of the patent application. The method of claim 10, wherein the polylactic acid-polyglycolic acid copolymer crosslinked alendronate is constructed as a polylactic acid-polyglycolic acid copolymer crosslinked alendronate three-dimensional scaffold (PLGA-ALN) -3D) or polylactic acid-polyglycolic acid copolymer cross-linked alendronate microspheres (PLGA-ALN-M). The method of claim 11, wherein the polylactic acid-polyglycolic acid copolymer crosslinked alendronate three-dimensional scaffold (PLGA-ALN-3D) has a pore diameter of 150 to 300 μm and an average of 85% porosity. The method of claim 11, wherein the polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALN-M) have a diameter of 50 to 100 μm. The method of claim 10, wherein the stem cells are human adipose stem cells (hADSCs). A method for enhancing differentiation of stem cells into a chondrogenic lineage, comprising culturing stem cells in a polylactic acid-polyglycolic acid copolymer having hyaluronan (HA) and the scope of claim 1 (1) In the microenvironment of PLGA-ALN). The method according to claim 15, wherein the polylactic acid-polyglycolic acid copolymer crosslinked alendronate (PLGA-ALN) is constructed to form a polylactic acid-polyglycolic acid copolymer crosslinked alendronate Ball (PLGA-ALN-M). The method of claim 16, wherein the polylactic acid-polyglycolic acid copolymer crosslinked alendronate microspheres (PLGA-ALN-M) have a diameter of 50 to 100 μm. The method of claim 15, wherein the stem cells are human adipose stem cells (hADSCs).