BMSCs are bone marrow-derived cells that play a key role in the renewal and regeneration of osteoblasts. BMSCs can differentiate into bone-forming osteoblasts and have been shown to be a primary source of osteoprogenitor cells. Moreover, BMSCs can be used as bone graft materials to treat bone defects. While the local osseous tissue is damaged by the pathogenic microorganism, BMSCs are activated and differentiate into osteoblasts to complete the repair of local bone dissolution. Failure of BMSCs to completely repair the local bone defects caused by infection may lead to local osteoporosis and even pathological fractures. Therefore, while using various antimicrobial agents to control infection, the consideration of the effect of drugs on osteogenic differentiation of BMSCs is crucial. Drugs with antibacterial properties and osteoinductive ability may play a better therapeutic role in orthopedic infections, such as osteomyelitis; whereas drugs that inhibit the differentiation of stem cells into osteoblasts and destroy the osteogenic microenvironment may adversely affect the repair of local osseous tissue. In this section, we will review the effects of different antimicrobial agents on osteogenic differentiation, and the overall situation is listed in Table 1.
Antituberculosis drugs: As a representative drug for the treatment of tuberculosis, rifampicin has a strong bactericidal effect on Mycobacterium tuberculosis. In addition, rifampicin has also been shown to exhibit anti-Gram-positive bacteria activity and kill the intracellular bacteria hidden in cells, and has a wide range of clinical applications. To demonstrate the potential toxicity of rifampicin and its effects on osteogenic differentiation of osteoblasts, researchers studied osteoblasts treated with different concentrations of rifampicin. The results showed that rifampicin did not cause toxicity to osteoblasts or affect the level of alkaline phosphatase (ALP) in the cells when the concentration of rifampicin did not exceed 10 μg/mL. However, when the drug concentration reached 100 μg/mL and above, the number of osteoblasts and intracellular ALP levels decreased significantly, and the decrease was over 75%. Another study demonstrated that rifampicin is cytotoxic to human bone marrow-derived MSCs at concentrations above 32 μg/mL and inhibited osteogenic diffe-rentiation potential of human bone marrow-derived MSCs in a concentration-dependent manner at concentrations ranging from 4-128 μg/mL. The collagen synthesis, mineralization effect, and expression levels of osteogenic genes in MSCs were inhibited to varying degrees with the increase in rifampicin concentration.
β-lactams: As a representative drug of β-lactam antibiotics, the discovery of penicillin has great significance in the history of human infectious diseases. It has been reported that penicillin, at a conventional blood concentration (30 μg/mL), does not inhibit the osteogenic differentiation process of human bone marrow-derived MSCs. When penicillin was added during the culture of human osteoblasts, cytotoxicity was observed when the penicillin concentration reached 500 μg/mL. At the same time, the differentiation function of osteoblasts was also significantly inhibited after penicillin concentration exceeded 500 μg/mL, and the intracellular ALP level was significantly decreased (above 75%) compared with the control group. Since penicillin cannot tolerate the enzymes produced by a variety of bacteria and is more likely to be destroyed, the probability of clinical drug resistance is increased and the clinical application is greatly limited. Therefore, some antibiotics that are artificially synthesized and can tolerate penicillinase are gradually replacing penicillin and play a greater role in the clinic. Both flucloxacillin and nafcillin are semi-synthetic peni-cillins that can tolerate penicillinase. It has been reported that flucloxacillin at conventional plasma concentrations (200 μg/mL) does not affect the osteogenic differentiation of human bone marrow-derived MSCs. Nafcillin can still exert its antibacterial effect under acidic conditions, but it has been reported that nafcillin has a strong inhibitory effect on the proliferation and differentiation of human osteoblasts. When its concentration exceeds 10 μg/mL, the ALP level in osteoblasts was drastically reduced.
Cephalosporins are an important branch of β-lactam antibiotics and play an important role in the treatment of various infectious diseases. Cefazolin, cefuroxime, cefotaxime, and cefepime are representative drugs of first, second, third, and fourth generation cephalosporins, respectively, and their effects on the differentiation of osteoblasts have been reported. Previous studies showed that cefuroxime does not alter the osteogenic differentiation of human bone marrow-derived MSCs at conventional blood concentrations (50 μg/mL). Cefazolin and cefepime cause osteogenic inhibition (above 25% and 75%, respectively) at concentrations up to 200 μg/mL, and cefotaxime inhibits the differentiation of osteoblasts (above 75%) at concentrations up to 500 μg/mL.
Carbapenems are a new class of β-lactams that are known for their broad spectrum. These drugs have strong antibacterial activity against most Gram-positive, Gram-negative, aerobic, anaerobic, and multi-drug resistant bacteria, and are one of the most important antibacterial drugs employed for the treatment of serious bacterial infections. Imipenem and meropenem are representative drugs that fall under in this category. Studies on the effect of these two drugs on differentiation of human osteoblasts have shown that imipenem does not have a significant effect on the differentiation potential of osteoblasts, while meropenem inhibits the differentiation of osteoblasts to a certain extent at concentrations of more than 500 μg/mL.
Macrolides: Macrolide antibiotics, drugs that inhibit bacterial protein synthesis by blocking peptide acyltransferase in bacterial ribosomes, are a class of drugs with extensive antibacterial spectrum. Azithromycin is a drug commonly used in clinical practice, and has certain inhibitory effects on various bacteria, mycoplasma, and chlamydia. Studies have shown that azithromycin does not produce cytotoxicity in the concentration range of 0-200 μg/mL; however, it inhibits the differentiation potential of osteoblasts at very low concentrations. When its concentration exceeds 10 μg/mL, the differentiation of human osteoblasts grown in the osteogenic induction environment was significantly inhibited, and the level of intracellular ALP synthesis decreased by more than 75%.
Aminoglycosides: Aminoglycoside antibiotics are a class of drugs that are effective against Gram-negative bacteria and aerobic bacteria, and gentamicin is a repre-sentative drug of this category. Studies have shown that gentamicin inhibits the osteogenic differentiation of human osteoblasts. When the drug concentration is less than 100 μg/mL, the drug does not have a significant effect on osteogenic diffe-rentiation. However, when its concentration exceeds 100 μg/mL, gentamicin exhibits osteogenic inhibitory effects. When its concentration exceeds 500 μg/mL, the osteogenic differentiation potential is almost completely suppressed. In another study, a similar phenomenon was observed in bone marrow-derived MSCs. When the gentamicin concentration reached 75 μg/mL, the proliferation and osteogenic differentiation activity of MSCs decreased significantly. In addition, studies have shown that gentamicin can inhibit the osteogenic differentiation of human bone marrow-derived MSCs in a dose-dependent manner within a concentration range of 50-200 μg/mL. The ALP level in the C2C12 cell line was similarly been reduced by gentamicin. Amikacin is a drug commonly used for the treatment of gentamicin-resistant infectious diseases. Its most prominent advantage is that it remains stable and active against the aminoglycoside inactivating enzymes produced by many Gram-negative bacilli. In addition, its effect on osteoblast differentiation is also less severe than that of gentamicin. At an amikacin concentration of 1000 μg/mL, the osteogenic differentiation of osteoblasts is still not significantly inhibited. Oste-ogenesis inhibition is exhibited only after the amikacin level reaches a very high concentration of 2000 μg/mL. As an aminoglycoside, tobramycin is often used for the treatment of gentamicin-resistant Pseudomonas aeruginosa infections. Studies have shown that tobramycin may have a lower cytotoxicity than gentamicin while exhibiting antibacterial effects. However, the effect of tobramycin on osteogenic differentiation is still inhibitory. When the concentration of tobramycin reaches 300 and 500 μg/mL, the osteogenic differentiation potential of human bone marrow-derived MSCs and osteoblasts is inhibited, respectively[14,20].
Tetracyclines: Tetracycline antibiotics exhibit a therapeutic effect on a variety of bacterial, rickettsial, chlamydial, and mycoplasma infections. Tetracycline is a representative member of such drugs. In addition to its role in killing various pathogenic microorganisms, tetracycline has been reported to exhibit bone tissue affinity and can, thus, be used for various targeted therapies. Studies related to osteogenic differentiation have shown that 10 μg/mL tetracycline can promote osteogenic differentiation of rat bone marrow-derived MSCs, increase ALP and mineralized nodules, and upregulate the osteogenic gene expression levels in MSCs. Doxycycline is the most commonly used tetracycline antibiotic, but unlike tetracycline, it exhibits a strong inhibitory effect on osteoblast proliferation and osteogenic differentiation. When its concentration reaches 100 μg/mL, the diffe-rentiation of human osteoblasts is severely inhibited. Minocycline is also widely used in clinical practice, and its antibacterial efficacy is relatively strong among tetracyclines. Similar to doxycycline, minocycline significantly inhibited the differentiation potential of osteoblasts (above 75%) at concentrations above 100 μg/mL.
Quinolones: Quinolones are a class of synthetic antibiotics that are widely used in a variety of clinical infectious diseases due to their excellent and broad-spectrum antimicrobial properties. Levofloxacin is a commonly used quinolone in the clinic. Studies have shown that it does not cause toxicity to human osteoblasts in the concentration range of 0-200 μg/mL, but when the drug concentration reaches 200 μg/mL or more, the differentiation potential of osteoblasts is significantly inhibited (above 75%). Ciprofloxacin is another representative drug of quinolones, which has poor biocompatibility and significantly inhibits the proliferation and differentiation of osteoblasts at concentrations above 10 μg/mL (above 75%).
Polypeptide antibiotics: Polypeptide antibiotics are a class of antibiotics with structural features similar to those of polypeptides, and their main members include polymyxins, bacitracins, and vancomycins. Colistin is one of the more commonly used polymyxin antibiotics. It mainly acts on Gram-negative bacteria and works synergistically with gentamicin. It has been reported in the literature that when the concentration of colistin reaches 100 μg/mL, the differentiation ability of human osteoblasts is inhibited. Bacitracin is a metal peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis; it can strongly inhibit Gram-positive bacteria and has antagonistic effects on the development of resistance to Staphylococcus aureus. Our previous studies have shown that bacitracin can promote the osteogenic diffe-rentiation of human bone marrow-derived MSCs in a dose-dependent manner, thus increasing intracellular ALP, collagen, and mineralization, and upregulating the levels of osteogenesis marker genes. When the concentration of bacitracin reached 100 μmol/L, its ability to promote bone differentiation decreased, but this effect was still stronger than that in the control group. Vancomycin is mainly used for the treatment of methicillin-resistant Staphylococcus aureus. There have been several reports on the effects of vancomycin on osteogenic differentiation. The general view is that vancomycin does not adversely affect the osteogenic differentiation of human osteoblasts and human bone marrow-derived MSCs at effective antimicrobial concentrations and higher concentrations[14,24,25]. However, it has also been reported that vancomycin inhibits the osteogenic differentiation of bone marrow-derived MSCs at a concentration of 200 μg/mL. Therefore, further research on the regulation of osteogenic differentiation by vancomycin needs to be conducted to determine whether the effect of this drug on osteogenic differentiation is related to cell type and drug concentration.
Other types of antibiotics: Metronidazole is a drug commonly used in the treatment of anaerobic infections in the clinic. Studies have shown that conventional plasma concentrations (20 μg/mL) of metronidazole do not affect the osteogenic diffe-rentiation potential of human bone marrow-derived MSCs. Trimethoprim (TMP) is a well-known sulfa drug enhancer with an antibacterial spectrum similar to that of sulfonamides. When TMP is combined with a sulfa drug, the combined antibacterial properties of both are greatly enhanced, and the formation of resistant bacteria can be reduced. Studies have shown that TMP does not affect the differentiation potential of osteoblasts in the concentration range of 0-200 μg/mL. However, when the concentration of TMP reaches 500 μg/mL, the osteogenic differentiation of the cells is inhibited. Linezolidone is a bacterial protein synthesis inhibitor and is a fully synthetic oxazolidinone antibiotic. The drug has good biocompatibility and does not affect the viability of osteoblasts between 0-500 μg/mL. However, when the concentration of linezolidone is greater than 10 μg/mL, osteogenic inhibition occurs. Salinomycin is a polyether antibiotic produced by Streptomyces albus. Studies have shown that 10 μM of salinomycin does not affect osteogenic differentiation and cellular mineralization of human bone marrow-derived MSCs.
In addition to the use of antimicrobial agents for the treatment of pathogenic microorganisms, which cause infection symptoms, activation of immune cells and secretion of some peptides with antimicrobial effects in the human body also play a decisive role in the elimination of infection. Lactoferrin is an important non-heme iron-binding glycoprotein found in milk, with powerful biological functions, such as broad-spectrum antibacterial, anti-oxidation, anti-cancer effects, and immune system regulation. It has been reported that lactoferrin promotes the differentiation of human adipose-derived stem cells into osteoblasts in a concentration-dependent manner and also promotes the expression of osteogenic genes. Similarly, other studies have found that lactoferrin promotes the proliferation of MC3T3-E1 osteoblast cells via the mitogen-activated protein kinase (MAPK) signaling pathway and promotes the differentiation of MC3T3-E1 into osteogenesis via the protein kinase A and p38 signaling pathways[28,29]. Hepcidin is a cysteine-rich polypeptide synthesized and secreted by the liver, which has a wide range of antibacterial and anti-protozoal functions. Studies have found that, in addition to regulating iron metabolism and antibacterial properties, hepcidin also regulates the function of rat bone marrow-derived MSCs. At a concentration of 0.2 mmol/L, hepcidin enhanced the mine-ralization ability of rat bone marrow-derived MSCs and upregulated the expression of osteogenic genes. The researchers found that this osteogenic differentiation may be related to the activation of the p38 signaling pathway. As an important part of the immune system, antimicrobial peptides (AMPs) can destroy microbial membranes and induce the death of pathogenic bacteria, having the potential to become a substitute for traditional antibiotics. The only natural antimicrobial peptide, cathelicidin (hCAP18/LL-37), was confirmed in 1995 and proved to exhibit antibacterial activity both in vitro and in vivo. Moreover, in addition to its resistance to pathogenic microorganisms, LL-37 has also been shown to promote the proliferation, migration, and osteogenic differentiation of rat bone marrow-derived MSCs. In the concentration range of 5-20 μg/mL, LL-37 promoted the osteogenic differentiation potential of MSCs in a dose-dependent manner. More importantly, LL-37 at a concentration of 10 μg/mL can reverse the osteogenic inhibition caused by lipopolysaccharide. However, since the peptide chain of LL-37 is too long and too difficult to synthesize, it is inconvenient to use it as a conventional therapeutic drug for bacterial infections and inflammatory diseases. Short-chain AMPs have recently attracted attention due to their lower production costs. Among the LL-37 active fragments of different lengths investigated, KR-12 is the shortest antimicrobial peptide with antibacterial activity. In our previous study, KR-12 stimulated oste-ogenic differentiation of human bone marrow-derived MSCs within an effective antimicrobial concentration (1-1000 μg/mL). This osteoinductive phenomenon also appears to be concentration-dependent.
Chinese traditional drug extracts
Chinese traditional drugs are mainly composed of botanicals (roots, stems, leaves, and fruits), animal drugs (viscera, skin, bone, organs, etc.,), and mineral medicines. Since such drugs are often present in a mixture rather than in a monomer form, their pharmacological effects are often studied by extracting the active ingredient of the drug. Similar to the above-mentioned antimicrobial drugs, some Chinese herbal extracts with antibacterial or anti-pathogenic properties have attracted a lot of attention in recent years[33-49]. Compared with traditional antibiotics, these Chinese traditional drug extracts exhibit less side effects and are less prone to drug resistance while exerting antibacterial effects. Among these herbal extracts, some promote osteogenic differentiation of bone marrow-derived MSCs, such as cordycepin, tanshinone, and baicalin[33-36,38,39]. If these extracts can exert stable antibacterial activity and simultaneously induce bone marrow-derived MSCs to differentiate into new osseous tissue by virtue of their osteoinductive properties, the clinical application prospects of these extracts will be more extensive. The Chinese traditional drug extracts that have been reported to regulate osteogenic differentiation and to exhibit antibacterial properties in recent years are also listed in Table 1.