Study On The Porous Composite Loaded With Rifampin And Its Application Potential In Bone Defect Of Osteoarticular Tuberculosis

Osteoarticular TB is the most common site of extrapulmonary TB, the incidence of which significantly and steadily increased year by year. However, it was unsatisfactory with the treatment in practice. It was proved that bone defects reconstruction and repair after debridement played a key role in surgical treating for osteoarticular TB. The reconstruction implants used in osteoarticular TB includes autogenous bone, allograft bone, and artificially synthesized biomaterials including calcium phosphate-based biocement materials, titanium mesh, and polymethylmethacrylate (PMMA). Unfortunately, they have not adequately met the needs of reconstruction requirements for osteoarticular TB due to a variety of reasons. It had great restricted the development of surgical treatment for osteoarticular TB. Therefore it is critically important to develop novel, ideal reconstruction materials for osteoarticular TB.Poly(D,L)-lactic acid (PDLLA) and nano-hydroxyapatite (nHA) are two most promising orthopedic reconstruction materials and core materials in drug delivery systems (DDS). They have been widely used in Medical research. Rifampicin (RFP) is a well-known and highly effective first-line anti-tuberculosis (anti-TB) drug. In the present study, a novel project was presented to address the difficulties in treating osteoarticular TB based on the guidance of the concept of translational medicine. A novel anti-TB reconstruction implant comprised of PDLLA and nHA in combination with RFP was firstly synthesized by solvent volatilization method. The characterization, degradation, drug release profile, biosafety and anti-TB capability were studied for synthetically evaluate the application potential of the composite for osteoarticular TB. The main thesis consists of five parts as below:1. Preparation and characterization of porous RFP/PDLLA/nHA composite. RFP, PDLLA and nHA were used as starting materials to fabricate a novel RFP/PDLLA/nHA composite by solvent evaporation method. It was spongiform and multiparous in structure. The production process were preliminary optimized by porosity, drug loading (DL), entrapment efficiency (EE) and shape controlling. The optimal proportion of three starting materials in wt.%is2:10:1. The porosity of the composite manufactured in this proportion is high to83.35±1.50%. The EE enable to be higher as76.77±0.82%. The DL is up to12.82±0.14%. X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) testified that the production process was steady and there were no generated impurities.2. Degradation and drug release of the porous RFP/PDLLA/nHA composite. The experiment in PBS indicated that the composite possess good degradation and excellent drug release profile. The walls of the holes collapsed and lots of degradation foraminulum (DF) formed during degradation process. The in vitro and in vivo weight loss ratios (WLR) of the composite were both higher than the pure pore PDLLA. After a transient burst release stage, it enable to sustained and steadily release for at least12w. The cumulative release percentage (CRP) is highly relevant to the WLR in vitro. There were a significantly liner relationship which could be fitted as Y=11.63+0.9549*X (R2=0.9836, SD=1.17, n=12).3. The biosafety of porous RFP/PDLLA/nHA composite. Porous RFP/PDLLA/nHA composite showed no dominant cytotoxicity but similar biocompatibility to MC3T3-E1cells with porous PDLLA/nHA composite. The histocompatibility was explored by means of implanting the composites into the subcutaneous tissues of KM mice. The results suggested the composites had excellent biocompatibility in vivo. The wounds healed well and the mice survived and grew well. Pathological results showed that it had not induced severe acute inflammation and rejection. The expressions of IL-6and TNF-a slightly increased in the early stage. However, it presented to follow a rapidly declining tendency.4. In vitro osteoinductivity of porous RFP/PDLLA/nHA composite. The in vitro osteoinductivity was preliminary evaluated by induction cultured with MC3T3-E1cells. The results showed that the alkaline phosphatase (ALP), osteocalcin (OCN) and collagen I (COL-I) of MC3T3-E1cells in RFP/PDLLA/nHA group expressed much more than the cells of RFP/PDLLA group. It indicated that the RFP/PDLLA/nHA composite possessed excellent osteoinductivity. It possessed excellent in vitro osteoinductivity for which enabled to facilitate pre-osteoblasts to be mature osteoblast (OB).5. The in vitro anti tuberculosis performance of porous RFP/PDLLA/nHA composite. Two composites (RFP/PDLLA/nHA, PDLLA/nHA) were cultured with M. tuberculosis in this study. The in vitro anti tuberculosis performance was correctly evaluated by combining with MicroMGITTM fluorescence reading system, acid-fast stain, auramine "o" stain and flow cytometry (FCM). The results showed that the former enabled to eliminate local M. tuberculosis by means of drug release. By contrast, the latter enabled to get along with M. tuberculosis friendly. In addition, it was testified that FCM is an excellent assessment method for biosafety and functionality evaluation for biomedical materials due to its ease of operation and high accuracy.These results proved that the porous RFP/PDLLA/nHA composite fabricated by solvent evaporate method possessed a great application potential for bone defect in osteoarticular TB. However, the treatment in the osteoarticular TB models in vivo remained to be further explored in the future. Meanwhile, the concrete influences on performance of the composite by the characteristic of starting materials are also needed to be studied. This study not only provide a novel option for osteoarticular TB treatment, but also raised of a novel design inspiration for the similar osteoarticular diseases in the future.