Tissue-engineered Bone With Vancomycin Delivery Fabricated By Decalcified Bone Matrix, Mesenchymal Stem Cells, And Controlled Release Alginate Beads Repairs Segmental Femur Defect

Background and Objective: At present, tissue-engineered bone (TEB) has a good repairing effect on bone defect and is one of treatments for bone defect. While bone defect and bone infection exist together, the implant of TEB associated with intravenous administration of antibiotic will be not suitable. The main reason is that the effective blood supply is hard to be formed between TEB and the host in a short time after implantation, which causes the few blood supply and ineffective antibiotic concentration in the implanted site, and the potential or present bacterium in implanted site can not be killed, and then propagation of bacterium will induce local infection and cause the failure of implantation. After polymethylmethacrylate (PMMA) beads containing antibiotic was used for treating bone infection and which got preonunced effect, the studies of local antibiotics delivery in bone tissue have aroused more concern and gained promising results. The current researches in this field focus on degradable biomaterials used by antibiotic delivery vehicles. Unlike PMMA, these biomaterials encapsulating an antibiotic do not require surgical removal, and which reduces the patients'pain. Therefore, aims of this study included:(1) Preparation and optimization of degradable vancomycin alginate controlled release beads. (2) The effects of vancomycin alginate beads on the proliferation and differentiation of mesenchymal stem cells (MSCs) will be evaluated when MSCs are cultured in the beads release solution. (3) The antibiotic concentration and osteogenesis in implanted sites will be assessed after TEB with optimized vancomycin alginate beads is implanted into a new goat model of segmental femur defect. Materials and Methods: (1) Vancomycin alginate beads were prepared by using the needle extrusion/external gelation method and coated by fibrin gel, the release of vancomycin from which was detected by high performance liquid chromatography (HPLC). (2) The activity of vancomycin released from beads was assessed according to the activity of standard preparation of vancomycin on the standard Staphylococcus aureus. And the difference of vancomycin activity between release and preparation was appreciated by an assay with methicillin-resistant staphylococcus aureus (MRSA) cultivated from clinical samples as a biological target. (3) MSCs derived from goat by density gradient centrifugation were identified by cell phenotypic profile and multilineage differentiation capacity. (4) The effects of vancomycin release solution on the proliferation and osteogenic differentiation of MSCs were assessed by the methods of growth curve, flow cytometry, immunohistochemistry staining, real-time RT-PCR, and western blot. (5) The proliferation of MSCs on DBM and survival of MSCs in vivo were observed by confocal laser imaging and scanning electron microscope after autogenic MSCs labeled by enhanced green fluorescent protein (EGFP) were seeded on decalcified bone matrix (DBM) or were implanted into goat bone defects. (6) After TEB with vancomycin delivery (fabricated by DBM, MSCs, and controlled release beads) was implanted into a new goat model of segmental femur defect, the local vancomycin concentration in implanted sites was determined by an assay of aspirate from the grafts. And the changes of creatinine and urea nitrogen were observed by an assay of venous blood from goats before and after implantation. (7) Three groups have been divided: TEB CONTROL, with TEB and without beads; BEADS CONTROL, with TEB and non-vancomycin beads; EXPERIMENT, with TEB and vancomycin beads. The osteogenic velocity and quality of the implants were assessed by the methods of X-ray score, Dual Energy X-ray Absorption (DERA), histopathologic and biomechanical methods. And the conditions of blood supply in implants were observed by radionuclide bone imaging and ink vessel perfusion.Results: (1) Vancomycin alginate beads were successfully formulated, and the content of vancomycin in beads was different due to the different concentration vancomycin solution and alginate solution, the maximum content was (27.35±0.90) % in 50 mg/mL vancomycin solution and 16% alginate solution. The maximum release days detected by HPLC were 23, which occurred in beads coated by 75 mg/mL and 90 mg/mL fibrin gel, respectively. (2) The minimum inhibitory concentration, minimum bactericidal concentration, and breakpoint sensitivity of vancomycin for S. aureus (ATCC 25923) were 1.2 mg/L, 2.1 mg/L and 5 mg/L, respectively. The beads coated by 75 mg/mL fibrin gel gave rise to effective concentrations through days 19 in accordance with breakpoint sensitivity. The same bactericidal effect on MRSA was taken in vancomycin preparation and vancomycin release solution. (3) The isolated cells were some adherent fibroblast-like cells. Through 3 passages, the percent of positive cells in CD29, CD44, CD62L, and CD45 were 98.70%, 99.54%, 1.10%, and 0.73%, respectively. Their osteogenic, chondrogenic, and adipogenic differentiation were determined by RT-PCR, immunohistochemistry staining, and special pathologic staining. (4) There were no significant differences between MSCs in release solution containing 15.00g/L or less concentration beads and MSCs in medium for assays of cell cycle and growth curve. Meanwhile, ALP activity, some osteoblast special mRNAs level, and some special osteogenic related proteins expression were increased in release solution (beads concentration≥10.00 g/L) compared with the control. (5) The results observed by confocal laser imaging and scanning electron microscope showed that most implanted MSCs could survive one month post-implantation and distribute in new bone trabecula four months post-implantation. (6) After TEBs were implanted in both-sides femur defects, beads with and without vancomycin were filled around TEB in right and left femur defects, respectively. The implanted site on the right (vancomycin beads) could stay bacteriocidal effectively for 21 days while the left (non-vancomycin beads), 0 day, and the blood, 2 days. There was no significant increase for creatinine and urea nitrogen between pre-operation and post-operation. (7) The Samantha X-ray score showed that there was no significant difference among TEB CONTROL, BEADS CONTROL, and EXPERIMENT 16 weeks after implantation. The difference in bone mineral density (BMD) value was similar to the Samantha score, which of EXPERIMENT group was (1.30±0.16)g/cm2. (8) The rate of limited torsion strength between operative femur (right) and normal femur (left) was (79.1±6.5)% in EXPERIMENT group, (80.2±5.1)% in TEB CONTROL group, and (77.1±1.8)% in BEADS CONTROL group, respectively. There was no significant difference in them. (9) There was no significant difference in the ratio of T/NT detected by radionuclide bone imaging between EXPERIMENT group and both control groups 4 and 8 weeks after operation. HE staining and ink perfusion showed that the vessels in all groups were reticulate distribution, regularly aligned and similar in diameter 16 weeks after operation. And there were some full-blown osteons in regeneration bone tissue section of all groups.Conclusion: (1) Preparing beads possessed the approving drug content and controlled release capability. And the release time was prolonged by fibrin gel coating compared with without coating. (2) The bioactivity of vancomycin from release solution was the same to that of vancomycin preparation. (3) MSCs from goat were gained according to the conventional methods. The beads release solution (beads concentration≤15.00g/L) did not affect the proliferation of MSCs and could promote osteogenic differentiation of MSCs. (4) The tracing used by retrovirus with EGFP gene was available to MSCs, which showed that implanted MSCs could survive in vivo and participate in new bone formation. (5) The local vancomycin concentration in implanted sites was effective and met the need of local anti-infection therapy after TEB was implanted into femur defect. The osteogenic quality of the implants in EXPERIMENT group was not affected by vancomycin beads. All suggested this system was suitable to treat the cases with both bone defect and infection.