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Description
Introduction
Bone grafting is among the most frequent forms of transplantation, utilized to manage a variety of bone defect cases. Enriching synthetic bone substitutes with immune-interacting properties can be a strategy to improve their performance. The host’s response to e.g. microbial components can result in both a positive or negative effect on bone formation. As an immunomodulatory strategy, we tested a variety of microbial-derived agents on their potential to enhance osteogenesis in biphasic calcium phosphate (BCP) scaffolds.
Materials and Methods
Implant preparation
BCP discs (6x9mm ø) comprising 65%-75% tricalcium phosphate and 25%-35% hydroxyapatite with a porosity of ±75% were used. Microbes tested included inactivated Staphylococcus aureus, Candida albicans, and Bacillus Calmette–Guérin (BCG) in a concentration of 105 -107units/ml. Microbial components tested included peptidoglycan, poly (I:C) high molecular weight, CpG oligodeoxynucleotid type C, Pam3CSK4, and curdlan at a concentration of 0.1µg/ml (low) and 1-10µg/ml (high). Microbial agents were diluted in phosphate buffered saline and combined with bone morphogenetic protein-2 (BMP-2, 5µg/scaffold), subsequently 200µl of the solution was loaded onto the scaffolds (n=10-12).
Animal Study
24 New Zealand White Rabbits (female, 4.5-5 kg) were included under the approval from the local Committee for Animal Experimentation. To pre-test the inflammatory response towards microbial agents, peripheral blood was collected, stimulated, and analyzed for the presence of IL-6 and IL-1β by ELISA. Scaffolds were implanted in subcutaneous pockets of the rabbits, with a control group (BCP with BMP-2) present in each animal. On week 2 and 3, fluorochrome labels calcein green and xylenol orange were administered. Fluctuations in body weight and temperature were monitored weekly. After a 5-week implantation period, samples were analyzed by histomorphometry (basic fuchsin/methylene blue) and fluorochrome imaging.
Results
Microbial agents stimulated a dose-dependent response in rabbit blood with increased levels of both IL-6 and IL-1β. As expected, the suboptimal dose of BMP-2 triggered moderate osteogenesis. Bone was present in only 17 of 23 rabbits, and a relatively high variation of bone area percentage was seen in the control group: 5%(7.9) Most of the microbial components tested showed an inhibiting trend towards bone formation, except for BCG and peptidoglycan. The mean differences (SD) in bone area percentage compared to the control were as follows; Staphylococcus aureus low -5%(8.1) and high -2.2%(6.1); Candida albicans low -3.2%(7.8) and high -4.1%(8.2); BCG low -0.7%(5.0) and high 0.3%(5.9); peptidoglycan low 2.3%(4.8) and high 3.9%(11.9); Poly(I:C) low -6.8%(9.6) and high -2.8%(7.4); CpG C low -2.8%(9.9) and high -0.9%(7.4); Pam3CSK4 low -2.1%(3.8) and high -0.4%(4.5); and curdlan low -2.7%(4.4) and high -2.7%(4.4). Interestingly for several groups, lower concentrations showed a stronger trend of hindering osteogenesis than higher concentrations.
Conclusion
The microbial agents can evoke an inflammatory response in rabbit blood. This response did not directly translate to effects on bone regeneration when such agents were loaded on BCP in the presence of suboptimal BMP-2 dosages. Further investigation is needed to determine the optimum range of concentrations in which microbial agents may have a beneficial effect on bone formation.
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