Preparation And Cytological Studies Of Thermo-Sensitive Hydrogels Loaded With PTH (1-34)

Background and Objective:Denture implanting has been widely applied in clinical field to repair the missingteeth for patients. The bone defects or osteoporosis around implants will affect rapidhealing of implants-osseointegration interface and long-term success rate. Improvingthe quality and quantity of bones has become the key to solve this problem.Parathyroid hormone (PTH), a drug for increasing the bone formation, can promotethe bone formation and increase the bone density by increasing the number andprolonging the lifespan of osteoblasts. So, it can increase the bone mass and improvethe osseointegration of the planting area. As a drug for the treatment of osteoporosis,PTH1-34has been approved by the U.S. FDA. In the past clinical trials and researchapplications as a means to promote bone formation and bone healing drugs, PTH weremainly administered by low-dose, intermittently, subcutaneously and systemically.However, the deficiencies of PTH, such as volatility and short half-life, limit itsfurther application in the field of Oral Implantology. Thus, it will bring broaderapplication prospects in the field of Oral Implantology to develop a delivery system toimprove the bioavailability of PTH for its good function to promote bone formation,.The temperature-sensitive hydrogel of poly (N-isopropyl acrylamide)(PNIPAAm) is currently the most widely studied temperature-sensitive hydrogel.According to the phase transition of the volume under different temperatures, it canrealize the slow releasing rate of the drugs. This study aims to synthesize thetemperature-sensitive hydrogel networks loading PTH1-34to achieve the controllablerelease of PTH1-34. Furthermore, by studying the effect of this system in promotingcell proliferation and differentiation in vitro, we can provide a theoretical guidance forfurther applications. Method:In this experiment, three temperature-sensitive biodegradable hydrogels withdifferent crosslinking degrees were synthesized via free-radical copolymerizationreaction using N-isopropyl acrylamide (NIPAAm) as the monomer, and abiodegradable polycaprolactone as crosslinker, and their structures were characterized.In addition, according to the application of different polycaprolactone crosslinkerproportion, we studied the swelling ratios under different temperatures, the drugloading efficiency and the drug release behaviors in vitro of the three PTH1-34loadedpoly (N-isopropyl acrylamide)(PNIPAAm) hydrogels. To evaluate the cytotoxic ofNIPAAm hydrogels, we fabricate PNIPAAm hydrogels without PTH1-34andpolyethylenimine(PEI), and they were co-cultured with HeLa cells, respectively. Weco-cultured PTH1-34with mouse osteoblast MC3T3-E1to select the optimalconcentration of PTH1-34for promoting cells’ proliferation and differentiation byMTT assay and alkaline phosphatase quantitative detection. According to the results,we co-culture PTH1-34loaded gels with MC3T3-E1to observe the capacity of thedrug-loaded gel system sustained releasing PTH1-34in vitro to promote MC3T3’sproliferation and differentiation by the MTT assay and alkaline phosphatasequantitative detection.Results:Three hydrogels with different crosslinking degrees were synthesized via radicalcopolymerization reaction taking N-isopropyl acrylamide(NIPAAm) as the monomer,and biodegradable polycaprolactone as the crosslinker. The hydrogels were marked asGel1, Gel2and Gel3.1. Because Gel2contained appropriate polycaprolactone crosslinker, when thetemperature changes from25℃to37℃, which is higher than Gel2hydrophobicshrinking, its pore size was the most appropriate for the release behavior of drug.After contracting, Gel1contained the largest volume and material aperture, and leadto the quick release of drugs, which was improper for the continuous release. Thesmallest contraction of Gel3lead to slowest release rate of the drug, which is notconducive to reach an effective concentration of the drug locally. The drug loadingefficiency of Gel1, Gel2, Gel3was0.241%,0.187%and0.138%, respectively. PTH1-34loaded in Gel2sustained released from Gel2for12days.2. Cytologic evaluation of PNIPAAm：(1) The emputy NIPAAm hydrogel group’s cell viability was up to90%,confirming that the material is non-cytotoxic, and it can be applied to subsequentexperiments and clinical studies.(2) By MTT assay and alkaline phosphatase quantitative detection, theconcentration of the intermittent administered PTH1-34to MC3T3which promotingthe most obviously cell proliferation was10-9mol/L.(3) By MTT assay and alkaline phosphatase quantitative detection, when thetotal concentration of PTH1-34loaded by Gel2is10-8,10-9mol/L, MC3T3–E1exhibited significantly effects promoting proliferation and differentiation ofcells（P<0.05）.Conclusion:1. Under the37°C conditions, the temperature-sensitive PNIPAAm hydrogelloading PTH1-34sustained released PTH1-34up to12days.2. The PNIPAAm hydrogel was non-cytotoxic, which also did not inhibitproliferation and differentiation of cells.3. The intermittent application of PTH can promote the proliferation anddifferentiation of osteoblasts, and the most appropriate concentration of PTH is10-9mol/L.4. Loading PTH1-34, the PNIPAAm hydrogel local sustained releasing effect cansignificantly promote the proliferation and differentiation of MC3T3-E1. It wasprompted that the temperature-sensitive hydrogel loading PTH1-34as a drug deliverysystem has a good potential application in the field of Oral Implantology, providingpossible solutions to solve clinical problems of insufficient amount of peri-implantbone.