Long-term And Localized Release Of BMP-2 Nanocapsule Used In Spinal Fusion

Background and ObjectBone healing, reconstruction of bone defect and spinal fusion related problems has long time been a focused area for orthopedic surgeons. Although autograft has been routinely used in bone fusion surgeries, there are still a lot of limitations and a new substituted method is needed. Marshall Urist first described bone morphogenetic proteins (BMPs),Wang et al obtained BMP-2 from bone tissue extract and its recombinant product in 1988. Thanks to the development of biotechnology, BMPs has been extensively studied and applied to clinical area. Numerous in vitro studies, animal experiments and clinical trials have proved the osteogenic ability of BMP-2. However, there are still limitations in BMP-2 use. As the rhBMP-2 protein is water soluble, it will be washed away quickly if directly used without a delivery material, and only 5% protein can remain. The half life of purified BMP-2 protein is very short, and it will be degraded within hours. Thus, to obtain a satisfactory result, BMP-2 is now being used in supra-physiological dosage. The market price for rhBMP-2 is quite expensive, consuming hundreds million US dollars per year. Moreover, if administrated in high dosage, it can also cause many side effects such as soft tissue edema, ectopic bone formation, inflammation reaction as well as immune reaction. In certain situation such as anterior cervical spine cases, it may result in paralysis and even fatal complications. An optimal delivery material is need for clinical use of BMP-2.Materials and Method1. Fabrication and Property Test of Long-term and Localized Release BMP-2 NanocapsuleFirst we tested the fabrication method on Bovine Serum Albumen (BSA),in search of the optimal parameters. According to the result, we chose to use positively charged monomer Spermine-V, with the ratio to neutral monomer 1:1, and the ratio of protein to monomer is 1:1000. We used the one-step in situ polymerization reaction to fabricate the nanocapsule. GDMA was prepared first, and AAm, Spermine-V as well as GDMA were added to the BMP-2 solution. NBMP-2 was harvest after hours of reaction. Small pieces of ACS were soaked in the mixed solution containing nBMP-2, sodium phosphate, EDC and NHS, and then the nBMP-2 was combined to ACS. Dynamic light scatter (DLS) was used to test the size and charge of nanocapsule. Matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF-MS) was used to check the combing reaction. Fluorescence microscope was introduced to test the difference of BMP-2, nBMP-2, and combined nBMP-2 washed away during the sponge swelling, to check the effect of combination. The reaction of nBMP-2 to basic PH value was also checked.2. In Vitro Test of Release Kinetics and Osteogenic Property of Long-term and Localized Release BMP-2 NanocapsuleSmall pieces of ACS were placed into Eppendorf tube. NBMP-2 was fabricated as described above and combined to ACS, basic buffer was added to dilute nBMP-2 to different concentration. BMP-2 solution, monomer Spermine-V and basic buffer was added to control tubes. Samples were collected at different time points. ELISA kit was used to test the quantity of BMP-2 to confirm the controlled release effect of nanocapsule on BMP-2. Alkaline phosphatase (ALP) staining kit and fluorescence kit were used to test the osteoinductive effect of different protein sample on BMSCs,C3H10T1/2 and MC3T3-E1 cells respectively.3. In Vivo Test of Long-term and Localized Release BMP-2 Nanocapsule Used in a Rat Spinal Fusion ModelThirty rats were allocated to three different groups according to the different implants. Group 1: nBMP-2+ACS; Group 2: BMP-2+ACS; Group 3: PBS+ACS. A middle line incision was made on the back of the rat at the segment of L4/5. The transverse process of L4 and L5 were then exposed and decorticated using a high-speed burr. The prepared implants were then placed flatly between L4 and L5 transverse process. After 8 weeks follow up, the rats were euthanized in CO2 tank, and the lumbar spine specimens were then harvested. X-rays were taken by using Faxitron Cabinet X-ray System and the specimens were also scanned by the SCANCO micro-CT System, evaluating the fusion rate and bone volume, to compare the osteoinductive ability of nBMP-2 and BMP-2 on rat spines.ResultAccording to DLS measurement, the average diameter of BMP-2 is 3.5 nm, and after the reaction, the diameters of nanocapsules were between 9nm and 12nm, averaged 11nm. The capsules were then inferred to be single or double layer molecules, and the thickness of the capsule should be between 1nm to 3 nm. The outer charge of these nanocapsules is +8.6 mV, which facilitates the reaction with ACS. Deposits were observed after the addition of EDC and NHS to BSA, which testified the feasibility of the combination reaction. According to MALDI-TOF-MS, the molar mass indicates single BSA protein before reaction, however, afterwards, it shows macromolecule molar mass, which again indicates the feasibility of the combination reaction. Under the fluorescence microscope, the fluorescence intensities of all the three samples were similarly high between each other. As time went by, all of them decreased, among which BMP-2 sample was quickest and almost disappeared after 1 hour. While nBMP-2 showed better maintenance of fluorescence intensity compared with BMP-2, which restored certain amount of fluorescence intensity after 11 hours. The combined nBMP-2 shows the best maintenance of fluorescence intensity, which preserved strong fluorescence intensity after 11 hours. It can be inferred that the combination reaction between the BMP-2 protein and the ACS are effective to combine them together. The nanocapsules were observed degrading after 3 hours and totally degraded after 6 hours when placed in basic environment, which indicates that nBMP-2 is PH value sensitive.2. ELISA test shows that the BMP-2 concentration of BMP-2 sample was highest at 0 point, quantified 433.9pg/ml. The concentration was then decreased rapidly to 148.9 pg/ml at 12 hours, and lower than 50 pg/ml after 3 days. The initial concentration of nBMP-2 sample was 144.4 pg/ml, lower than BMP-2 solution, however, did not decrease afterwards. After 14 days, it slightly decreased to 156.7 pg/ml, and at 21 days, it still remained around 89.9 pg/ml. The ALP staining test showed at 0 point, the ALP activity of BMP-2 was higher than nBMP-2, after 12 hours, ALP activity tested in all three different cells shows nBMP-2 slightly higher than BMP-2 samples, which turned to be significant after 3 days, and lasted until the ending point 21 days. The result of ALP fluorescence test is in accordance with the staining test. The ALP activity of both monomer and basic buffer kept to be at low level and did not show difference between each other.3. X-ray images shows eight weeks post-operatively, 3 rats got bilateral fusion, while 4 unilateral fusions and 3 non fusions. All the rats in nBMP-2 group got satisfactory bilateral fusion. In the PBS buffer group, non of the rats showed any new bone formation except two of them, which showed some evidence of unilateral fusion. Manually tested fusion rate of BMP-2, nBMP-2 and PBS Buffer group was 40%, 100% and 0 respectively. The micro CT scan got similar result. The average bone volume of BMP-2 was 221.0±22.8 mm~3, which is significantly higher than PBS Buffer group (P=0.001). The average bone volume of nBMP-2 group is 269.2±18.0 mm~3, higher than the other two groups.ConclusionIn this study, we fabricated a new kind of nanocapsule by using positively charged monomer Spermine-V and crosslinker GDMA, which is nano-grade in size and can cover the surface of BMP-2 protein. Then we combined the nBMP-2 protein to ACS by polymerization reaction. We carried out several different in vitro and in vivo experiments to test the effect of this kind of new nanocapsule. It shows that the covalent bond of nBMP-2 to ACS significantly decreased the lost of protein. The utilization of basic environment as driving force of BMP-2 protein release makes it possible to get long-term controlled release kinetics, which enables the maintenance of high concentration of BMP-2 around the intended area and thus a higher fusion rate. Before release, the surface of BMP-2 is covered by single layer protein, and the active groups are protected inside the capsule, the BMP-2 protein is protected from degradation. Also, as the groups are hided inside the capsule, it can avoid inflammation reaction and immune reaction. Thus, theoretically, this new type of nanocapsule satisfies the need of an ideal BMP-2 delivery system, such as biocompatibility, predictable biodegradability, low immunogenicity and antigenicity, controlled and metered/tailored protein release, malleability and ease of manufacture aafety, stability, sterility, availability and cost-effectiveness. However, more in vitro and in vivo experiment is needed to testify the low immunogenicity and antigenicity property.