Sage Journals: Discover world-class research

Abstract

Background

Prosthetic-joint infection (PJI) is one of the severest complications after arthroplasty. However, antibiotics are not effective in the bacteria in biofilm outside the prosthetic-joint. Antimicrobial peptides have an efficient antimicrobial activity in staphylococcus aureus compared with conventional antibiotics.

Methods

Bone marrow stem cells (BMSCs) were isolated, cultured and transfected with cathelicidins antimicrobial peptides proline-arginine-rich 39 amino acid peptide (PR-39) lentivirus. The expression of PR-39 gene in BMSCs was detected by RT-PCR, and the antibacterial activity of PR-39 was measured by agar diffusion method. The transfection efficiency was detected by fluorescence microscopy. The infection model of artificial knee joint in rabbits were established. Kirschner wire was used as the knee joint implant to implant the distal femur through the femoral intercondylar fossa of rabbits. 24 rabbits were randomly divided into 2 groups for the above operations: group A was inoculated 0.5 mL into the joint cavity immediately after the incision was sutured 1 × 10⁷ Staphylococcus aureus of colony forming unit (CFU), group B was inoculated with Staphylococcus aureus and PR-39. After operation, the wound conditions and histological changes were observed by X-ray and optical microscope respectively, CRP and erythrocyte sedimentation rate were measured by test assay.

Results

The transfection efficiency of lentivirus vectortransfected BMSCs was 74.09%. The supernatant of lentivirus vector had obvious inhibitory effect on Staphylococcus aureus, and the antibacterial rate was 98.43%. 100% infection observed in group A while few infection observed in group B; serum CRP and ESR at a high level in group A while decreased in group B after operation. There were no significant difference in CRP and ESR between the pLV/PR-39 group and pLV/EGFP group at day 1 and 3 respectively after surgery. However, CRP and ESR in the pLV/PR-39 groupwere significantly lower than the pLV/EGFP group at day 7 and 14 respectively after operation.

Conclusions

Rabbits planted BMSCs expressing PR-39 were significantly increased resistance to Staphylococcus aureus in PJI than control group thus showing great potential for preventing implant-associated infection. It will provide a potential new therapeutic agent for implant-associated infection.

Keywords

PR-39 antimicrobial peptide infection prosthetic joint biofilm bone marrow stem cells

Introduction

Infections associated with prosthetic joints are the serious complications of arthroplasty. Although the use of perioperative antimicrobial prophylaxis and a laminar airflow surgical environment have reduced the risk of intraoperative infection to less than 1% after hip and shoulder replacement and to less than 2% after knee replacement,^1,2,3 the increase of patients subjected to arthroplasty, the absolute number of prosthetic-joint infection (PJI) still not decrease. It was reported that the increase of PJI is more than 4000 in patients received total hip replacement in American every year.⁴ The microorganisms growing on the surface of prosthetic-joint, which are up to 1000 times more resistant to growth-dependent antimicrobial agents than their in vitro counterparts.⁵ The patients usually were required surgical and prolonged medical management.

Recently, it was revealed that inhibited the increasing of Porphyromonas gingivalis on the surface of titanium and promoted the differentiation of osteoblastic cells in vivo by immobilizing cationic antimicrobial peptide to titanium.⁶ We hypothesized that antimicrobial peptide is a potential and promising therapeutic agent for implant-associated infection. Cathelicidins are cationic antimicrobial peptides with broad-range antimicrobial activity against bacterial, viral and fungal pathogens.⁷ Cationic antimicrobial peptides are widespread in nature and play an important role in innate immunity which exist in humans, mice, cows, pigs and other rodents. Proline-arginine-rich 39 amino acid peptide (PR-39) is a proline-arginine-rich cationic antibacterial peptide isolated from pig intestine.⁸ Inhibition of the PR-39 activation promoted bacterial growth in pigs.⁹ PR-39 can also decrease inflammation and promote wound repair.^10,11 We thus hypothesized that PR-39 might be used to protect against implant-associated infection of orthopedic.

It is difficult and expensive to produce antimicrobial peptides in vitro. Moreover, they are sensitive to protease digestion in vivo. Therefore, persistent expression of antimicrobial peptides in the areas of infection by the method of genetic engineering is urgent. The purpose of the experiment is to verify the preventive effect of antibacterial peptide PR39 on periprosthetic infection, which may provide a new solution for the treatment of periprosthetic infection in the future.

Materials and methods

Ethical statement

The study protocols was approved by Ethics Committee of the Chongqing Medical University (permit number: 2020-015).

Cathelicidin lentivirus production

To construct PR-39 expression plasmid pGC-FU/PR-39, PR-39 fragment containing a whole coding region of PR-39 gene was amplified from the plasmid of pBluescript II SK-PR-39 (provided by Dr. Daniela Tirziu, Dartmouth Medical School, USA) and inserted into enhance green fluorescent protein (EGFP) tagged expression vector pGC-FU (Genechem) to construct PR-39 expression plasmid pGC-FU/PR-39.¹² Moreover, enhancer sequence was inserted into the front of PR-39 gene to enhance the expression of target gene. The recombinant PR-39 lentiviruses (pLV/PR-39) and empty lentiviruses with EGFP gene (pLV/EGFP) were produced by cotransfecting 293T cells by use of lipofectamine 2000 (Invitrogen) according to standard protocols. The mixture of packaging plasmids pHelper 1.0, pHelper 2.0 and pGC-FU/PR-39 were cotransfected into human embryonic kidney cells (293T cells) by use of lipofectamine 2000 (Invitrogen) in serum-free medium. After 24 h, the medium was replaced with complete medium, and cells were further incubated for 48 h. Viral supernatant was collected, filtered (0.45 μm filter) and concentrated as described previously.¹³ The empty lentiviruses with EGFP gene (pLV/EGFP) was also generated with the same methods as a negative control. Viral stocks were stored at −80°C refrigerator.

Isolation, cultivation and transduction of bone marrow stem cells

Rabbit bone marrow stem cells (BMSCs) were separated following Friedenstein’s protocol.¹⁴ The Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, GIBCO) media was changed every 3 days. Cells passage was cure when cells grow to 90% confluence. For transduction with viral stocks, the passage 3 BMSCs were grown in six-well plates with the plenty of 1 × 10⁵/well overnight. Before infection, the medium was replaced by serum-free medium supplemented with Polybrene (8 μg/ml) and viral stocks at the multiplicity of infection (MOI) 10, cells were incubated at 37°C overnight. The empty vector pLV/EGFP infected BMSCs as the control group. The medium was replaced with a fresh medium without Polybrene on the second day. After expansion in culture for 72 h, BMSCs were observed with fluorescent microscope, and the transfection efficiency was analyzed by detection of enhanced green fluorescence protein with Flow Cytometer.

Expression of cathelicidin antimicrobial peptide PR-39 in bone marrow stem cells

Total RNA was isolated from infected pLV/PR-39 after 72 h, and the BMSCs were harvested. Total RNA was prepared with Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was conducted according to the RT-PCR kit protocol (Takara). Reverse transcription was performed using 5 μg of total RNA in 10 μL of the following solution: 2 μL MgCl₂, 1 μL 10× RT buffer, 1 μL dNTP, 0.25 μL RNase inhibitor, 0.5 μL AMV reverse transcriptase, 0.5 μL oligo dT primer and 3.75 μL RNase free ddH₂O. The reaction was incubated at 30°C for 10 min, 42°C for 50 min, 95°C for 5 min, and 4°C for 5 min 5 ng cDNA was added 25 μL reaction volume. PCR reactions used two primers (5′-CACTGTGGCTTCTGCTGC-3′, 5′-GATGGGTTCAAGGTGACTG-3′) for PR-39 and two primers (5′-TCACCATGGATGATGATATCGC-3′, 5′-CGTGCTCGATGGGGTACTTCA-3′) for the housekeeping gene β-actin as a reference.¹⁵

The BMSCs infected with lentivirus vector were harvested and lysed with cold cell lysis buffer. Protein concentration was determined with the bicinchoninic acid (BCA) protein assay kit (HyClone, Pierce) according to manufacturer’s instructions. Proteins were separated following the methods,¹² and transferred onto nitrocellulose membranes. The membrane was blocked with 10% nonfat dry milk 0.1% Tween 20 in Tris-buffered saline, pH 7.6 at room temperature for 3 h, incubated at 4°C overnight with mouse anti-GFP antibody (Santa Cruz) (dilution 1:1000), and then incubated at room temperature for 1 h with goat and with enhanced chemiluminescence (Amersham) detection.

PR-39 antimicrobial activity assays

To examined the efficacy of pLV/PR-39-modified BMSCs on bacteria, the supernatant of pLV/PR-39-modified BMSCs and pLV/EFG-modified BMSCs was collected and condensed with the freeze dryer (purchased from SIM Company, USA) in the ratio of 10:1. 20 sterile cuvettes with 2 mL tryptic soy broth (TSB) (Sigma) were divided randomly into A, B, C and D groups respectively. There were 5 cuvettes in every group. Staphylococcus aureus (no: ATCC25923) were inoculated in every cuvettes except group A. The inoculums were adjusted for each organism to yield a cell concentration of 5 × 10⁵ colony forming units (CFU)/ml,¹⁶ according to previously standardization by the National Committee for Clinical Laboratory Standards (NCCLS, 1990). A total of 0.1 mL condensed supernatant of pLV/EGFP-modified BMSCs were added into every cuvettes of group C as a control. 0.1 mL condensed supernatant of pLV/PR-39-modified BMSCs were added into every cuvettes of group D. Group B was a negative control (cuvettes with broth and the inoculums). After incubated at 37°C for 24 h in aerobic medium, bacterial counts were determined by optical density (OD) at 600 nm.^12,17 Before monitoring, the zero adjustment of instrument was operated with broth of group A to decrease the error. The inhibitory percentage of PR-39 was calculated according to the following equation¹⁸:

Inhibitory percentage (%)=(1-absorbance group D/absorbance group B)×100%

CFU/ml was determined after plating samples overnight on tryptic soy broth agar.

Establish the PJI model of rabbit

The 3 passages BMSCs infected with lentivirus were harvested in the concentrations of 1 × 10⁸/ml with PBS. Infected joint replacement models have been successfully established as described by Sheehan et al..¹⁹ A total of 24 New Zealand white rabbits (2.5–3 kg) which were provided by the experimental animal center of Chongqing Medical University were randomly divided into two groups. Sedation was achieved using a weight dependant dose of phenobarbital 30 mg/kg intravenous. Animal were observed for 5 min until anaesthesia was effective, and were monitored by a veterinary technician. After shearing hair of the knee. The lower extremities of each animal were draped with sterile surgical drapes and secured with towel clips. The skin over the medial aspect of knee was incised, and drill hole in the local of femoral intercondylar notch of rabbits. 2 × 10⁵(0.2 mL)BMSCs infected with pLV/PR-39 were injected into the intramedullary of femora of the test group (group A). The BMSCs infected with pLV/EGFP were injected into the second group as a negative control (group B). The 2 mm kirschner wire (15 mm long) were inserted into the intramedullary of femora instantly. All surgical procedures were performed under sterile conditions. Intramedullary implantation of the metals was chosen as this closely mirrors the human situation of arthroplasty and long bone internal fixation surgery. 100 µl (equivalent to 4 × 10⁶ colony forming units) of Staphylococcus aureus was inoculated followed in joint.²⁰ It was designed to model acute infection after artificial joint replacement. To standardize the surgical trauma, all the operations were done by the same person. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were monitored in 1 d, 3 d, 7 d, 14 d respectively by test assay after surgery. Nephelometry was performed in the detection of serum CRP. Anti-CRP antibody was put into blood, then optical density of blood detected by automatic biochemistry analyzer. Automatic erythrocyte sedimentation rate analyzer was used to detect the ESR according to the manufacturer’s instructions. 2 weeks later, phenobarbital was injected intravenously to anesthetize animals. Femoras were cut down 20 mm at the supra femoral intercondylar notch under aseptic techniques, and metals were removed. Femoras were placed into sterile container. Choose one specimen of each group to observe by histological examination. They were brought into 4% paraformaldehyde solution for 24 h and subsequently sections were cut using the grinding-technique and stained with hematoxylin-eosin-azureⅡwhich was described elsewhere.^21,22 Other specimens were weighed, grinded and diluted in normal saline (1 mL/g), homogenized under sterile conditions using a tissue homogenizer as the describe of Isiklar et al..²³ Tenfold dilution of bone homogenates was made, and 1 mL aliquots were plated into tryptic soy broth agar plates. Colony forming units were quantitated after incubation at 37°C for 24 h. Both euthanasia methods and disposition of animal’s body at the end of study were performed according to the animal research: reporting of in vivo experiments (ARRIVE) guidelines. On the 14_th day after the operation, the animals were fixed on the cardboard and the right knee was taken in the straight position for X-ray (completed by the Radiology Department of the First Affiliated Hospital of Chongqing Medical University).

Turnover of bone marrow stem cells in vivo

To observe the turnover of BMSCs in vivo, 2 × 10⁵ pLV/EGFP-modified BMSCs were implanted under the tibia periosteum of rabbits. Four weeks later, lump was observed on the tibia. It was analyzed with histopathological detection.

Histopathological examination

Animals were finally euthanized 14 days after operation, the lower end of the right femur was taken 1.5 cm, the implants were taken out, and fixed with 10% polybasic formaldehyde for 24 h. Put the bone tissue removed above into the decalcifying solution at room temperature, and replace the decalcifying solution every 4 h until the pin can penetrate the bone dense substance; Paraffin embedded, continuous sectioning, with a thickness of 10 μM thick slice. Bake the slices overnight in a 60°C constant temperature oven. 10 μM thick slice sections were cut and stained with hematoxylin and eosin (H&E) for general pathological examination. The pathological changes were observed by a microscope and photographed at 200× magnification.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical comparisons among the three groups were evaluated using analysis of variance (ANOVA), followed by post-hoc Dunnett’s t-tests. Statistical comparisons between the two groups were evaluated using the student t test. Differences in values were considered significant or extremly significant when p < 0.05^* or p < 0.01^**.

Results

Lentivirus vector were produced and transducted into bone marrow stem cells

The lentivirus vector pLV/PR-39 was produced in 293T cells with the three plasmids of lentivirus. BMSCs were separated successfully with explantation of bone marrow. 72 h after transduction of viral stocks, expression of EGFP was detected with fluorescence microscope. The efficiency of transduction detected with Flow Cytometer was approximately 74.09%.

Expression of PR-39 in bone marrow stem cells

72 h after infected pLV/PR-39, the expression of the mRNA encoding the full-length PR-39 was identified by RT-PCR. Expression of the mRNA encoding the full-length PR-39 was verified by using RT-PCR. The flow chart of the experiment was presented in Figure 1.

Figure 1.

Flow chart of the experiment.

Antimicrobial activity of PR-39 against Staphylococcus aureus in vitro

After incubation at 37°C for 24 h, broth in group A and D was clear. In additon, broth was turbid in group B and group D. Antimicrobial activity was indicated by the lack of turbidity or color change in the medium and quantitated through the absorbance (Figure 2(a)) and CFU (Figure 2(b)) respectively. Compared with group B, cultivation supernatant of pLV/PR-39-modified BMSCs displayed excellent antimicrobial activity against Staphylococcus aureus (p < 0.05). The inhibition percentage (%) was approximately 98.43%, whereas compared with group B, collected cultivation supernatant of BMSC infected empty vector had no effect on the growth of Staphylococcus aureus compared with group B (p > 0.05).

Figure 2.

PR-39 antimicrobial activity assays. (a) The mean optical density (OD), (b) Colony forming units (CFU). The data represent the mean ± SD. ^**p < 0.01 vs. group B and group C.

The protective effect of additional cathelicidin antimicrobial peptide PR-39 in orthopedic bacterial material infection

One rabbit died for a noninfectious cause after surgery, and two rabbits showed clear purulent infections signs such as sinus tract in the control group, whereas wound healing was observed in all rabbits of PR-39 group. Serologic examination (ESR, Figure 3(a); CRP, Figure 3(b)) confirmed that PR-39 significantly palliated the systemic inflammatory reaction. But there was no difference in histological examination of inflammatory corpuscles between the two groups, and a large number of leukocytes infiltrated in both groups. It may be attributed to neutrophil chemotactic role of PR-39.²⁴ And other phenomena previously observed in PR-39, like angiogenesis²⁵ was also absent. Otherwise the difference was verified by microbiology methods. To test the resistance to infection in pLV/PR-39-modified rabbits, bacteria from bone samples were isolated and plated. It was revealed that the colony forming units (CFU) of PR-39 group significantly decreased than that of control group (p < 0.01) (Figure 4).

Figure 3.

Inflammatory markers in infected rabbits. (a) The mean erythrocyte sedimentation rate (ESR), (b) The mean C-reactive protein (CRP). The data represent the mean ± SD. ^*p < 0.05 vs. pLV/PR-39 group.

Figure 4.

The CFU in each Gram of bone tissue. The data represent the mean ± SD. ^**p < 0.01 vs. pLV/PR-39 group.

Osteogenic of BMSCs in vivo

To investigate the turnover of BMSCs which planted in vivo, BMSCs infected pLV/PR-39 were planted under the tibia periosteum of rabbits (Figure 5(a)). 4 weeks after surgery, it was found that there were lumps on the tibias, and there was bone trabecula presenting green fluorescence under the fluorescent microscope. On the contrary, green fluorescence wasn’t observerd in the soft tissue (Figure 5(b)–(e)). It suggested that the BMSC infected pLV/PR-39 differentiated into bone tissue.

Figure 5.

Turnover of BMSCs in vivo. (a) 4 weeks after implanted pLV/EGFP-modified BMSCs under the tibia periosteum of rabbits, there was a lump on the tibia. Frozen section of this lump was made. (b) Bone trabecula was observed under a microscope in bright field×100. (c) Bone trabecula observed under a microscope with green fluorescence in fluorescent field×100. (d) Bone trabecula observed under a microscope in HE staining×100. (e) Bone trabecula observed in green fluorescence×100.

Imaging results in vivo

Radiographs taken 14 d after surgery showed that all the built-in keratophores were well positioned, their borders were blurred, there were X-ray translucent bands around them, and the lower femur had reduced bone density and mild osteoporosis (Figure 6(a)). The borders of the uninfected ones were clear and the bone quality was better (Figure 6(b)). In the right knee orthopantomogram of rabbits taken 14 d after surgery, all animals in both groups had good positions of the built-in material in the femoral end of the right knee, and in the experimental group, the boundary of the built-in material was clear and the bone density of the lower femur was mildly increased, while in the control group, the boundary of the built-in material was blurred and there was an X-ray translucent band around it, and the bone density of the lower femur was reduced and there was mild osteoporosis (Figure 6(c), Figure 6(d)).

Figure 6.

(a) X-ray of the uninfected implant. (b) X-ray of the infected implant 14 d after operation. (c) X-ray of planted the BMSC infected pLV/PR-39. (d) X-ray of unplanted the BMSC infected pLV/PR-39.

Histological observation in vivo

Light microscopy showed a large infiltration of inflammatory cells around the infected endophyte (Figure 7(a)), while no inflammatory cells or erythrocytes were seen in the uninfected ones (Figure 7(b)). The decalcified bone sections of the two groups of animals showed a large number of inflammatory cell infiltrates around the endophytic tissue under light microscopy, and no significant differences were seen (Figure 7(c), Figure 7(d)).

Figure 7.

HE staining results. (a) Microscopic organization around the infected implant via HE staining×200. (b) Microscopic organization around the infected implant via HE staining×200. (c) Microscopic organization around the implant with planted the BMSC infected pLV/PR-39 via HE staining×200. (d) Microscopic organization around the implant without planted the BMSC infected pLV/PR-39 via HE staining ×200.

Discussion

In orthopedics, because of the implant, bacteria could not be handled radically.^{5,26,27,28,29} Therefore, infection can only be eradicated by traumatic removal of implants and continuous intravenous antibiotics. It is hard for patients. Fortunately, Makihira et al.⁶ revealed that the implant-associated infection could be prevented through immobilizing antimicrobial peptide on the surface of implants.

Antimicrobial peptides have the roll of natural immunity in vivo which were found in some animals such as pig. Previous study found that PR-39 is a cationic antimicrobial peptide isolated from pig intestine.³⁰ Antimicrobial peptide PR-39 have broad range of antimicrobial activity,^31,32,33 which may induce expression of syndecan-1 in wound repair³⁴ and cut down tissue damage during inflammation.³⁵ However, the shortage of antimicrobial peptide such as high cost and protease digestion in vivo limits its clinical application. Therefore, we infected PR-39 gene into rabbit BMSCs to prevent PJI. In the present study, BMSCs could express PR-39 in a long term because of the PR-39 gene conforming in the DNA of cells with lentiviral system. It was showed that the pLV/PR-39-modified BMSCs implanted in the medullary space of femora could secrete the PR-39 in a long trem in vivo to provide persistent protection against prosthetic-joint infections. PR-39 group had better healing of operative incision and slighter inflammatory reaction than negative control in our study. Compared with the control group, the experiment group showed significant difference in ESR and CRP at the 7 days and 14 days respectively after operation. We also found that the differences in CRP data at 14 days was not significant as long as the CFU. It may be interpreted that surgical trauma effect CRP data. Clinical research showed that the CRP data of patients was still higher than normal level in 14 d postoperative.³⁶

As for the strategy of BMSCs gene expression, pLV/PR-39-modified BMSCs could express the propeptide consisting of signal peptide and mature peptide in our study. Due to the guiding of signal peptide, the mature peptide could be secreted out of the cells. Therefore, the supernatant of pLV/PR-39-modified BMSCs in vitro could inhibit bacteria in broth. Although the nephrotoxicity and hepatotoxicity of PR-39 hasn’t been reported in this study, the long-term expression of PR-39 in vivo may be harmful to body, or pLV/PR-39-modified BMSCs may different into tumor after a longer time. In the study, we found that this worry was unnecessary. We planted pLV/PR-39-modified BMSCs under the tibia periosteum of rabbits. 4 weeks latter, the BMSCs differentiated into bone tissue. These results suggested that the BMSCs planted into the medullary space of femora could be stimulated mineralization. It avoids the potential side effects of over-expression of exogenous gene and spontaneous mutation of BMSCs. It was revealed that the strategy of genetic engineering is safe which give us a great potential method for preventing prosthetic-joint infections. It was found that antibacterial peptide PR-39 can be successfully transfected into rabbit BMSC and expressed successfully. The expressed PR-39 has strong antibacterial effect through in vitro and in vivo experiments, and can effectively prevent insertion-related infection, which may provide a new idea for the treatment of artificial joint infection.

Footnotes

Acknowledgements

Anonymous ...

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Scientific research and seedling breeding project of Chongqing Medical Biotechnology Association (cmba2022kyym-zkxmQ0003) and 2022 scientific research project of Chongqing Medical and Pharmaceutical College (ygz2022104).

ORCID iD

Jingxin Mao

References

Thornley

Evaniew

Riediger

, et al. Postoperative antibiotic prophylaxis in total hip and knee arthroplasty: a systematic review and meta-analysis of randomized controlled trials. CMAJ Open 2015; 3(3): 338–343.

Sperling

Kozak

Hanssen

,et al. Infection after shoulder arthroplasty. Clin Orthop 2001; 382: 206–216.

Tande

Patel

. Prosthetic joint infection. Clin Microbiol Rev 2014; 27(2): 302–345.

Egrlich

Stoodley

Kathju

, et al. Engineering approaches for the detection and control of orthopaedic biofilm infections. Clin Orthop Relat Res 2005; 437: 59–66.

Stewart

William Costerton

. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358: 135–138.

Makihira

Shuto

Nikawa

, et al. Titanium immobilized with an antimicrobial peptide derived from histatin accelerates the differentiation of osteoblastic cell line, MC3T3-E1. Int J Mol Sci 2010; 11(4): 1458–1470.

Meyerholz

Ackermann

. Antimicrobial peptides and surfactant proteins in ruminant respiratory tract disease. Vet Immunol Immunopathol 2005; 108(2): 91–96.

Holani

Shah

Haji

, et al. Proline-arginine rich (PR-39) cathelicidin: Structure, expression and functional implication in intestinal health. Comp Immunol Microbiol Infect Dis 2016; 49: 95–101.

Cole

Shi

Ceccarelli

, et al. Inhibition of neutrophil elastase prevents cathelicidin activation and impairs clearance of bacteria from wounds. Blood 2001; 97: 297–304.

10.

Zhou

. Apidaecin-type peptides: biodiversity, structure-function relationships and mode of action. Peptides 2006; 27(9): 2350–2359.

11.

Zhu

Gao

. A fossil antibacterial peptide gives clues to structural diversity of cathelicidin-derived host defense peptides. FASEB J 2009; 23(1): 13–20.

12.

Lee

Ohtake

Zaiou

, et al. Expression of an additional cathelicidin antimicrobial peptide protects against bacterial skin infection. Proc Natl Acad Sci USA 2005; 102(10): 3750–3755.

13.

Lois

Hong

Pease

, et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 2002; 295: 868–872.

14.

Friedenstein

Chailakhyan

Gerasimov

. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 1987; 20(3): 263–272.

15.

Yang

Wang

, et al. Cyclin L2, a Novel RNA Polymerase II-associated Cyclin, Is Involved in Pre-mRNA Splicing and Induces Apoptosis of Human Hepatocellular Carcinoma Cells. J Biol Chem 2004; 279: 11639–11648.

16.

Carson

Hammer

Riley

. Broth micro-dilution method for determining the susceptibility of Escherichia coli and Staphylococcus aureus to the essential oil of Melaleuca alternifolia (tea tree oil). Microbios 1995; 82(332): 181–185.

17.

Domínguez-Bello

Pacheco

Ruiz

, et al. Resistance of rumen bacteria murein to bovine gastric lysozyme. BMC Ecol 2004; 4: 7.

18.

Kil

Seong

Ghimire

, et al. Antioxidant and antimicrobial activities of crude sorghum extract. Food Chemistry 2009; 115(4): 1234–1239.

19.

SheehanMcKenna

Mulhall

Marks

, et al. Adhesion of Staphylococcus to orthopaedic metals, an in vivo study. J Orthop Res 2004; 22: 39–43.

20.

Arens

Schlegel

Printzen

, et al. Influence of Materials for Fixation Implants on Local Infection: An Experimental Study of Steel Versus Titanium DCP in Rabbits. J Bone Joint Surg Br 1996; 78(4): 647–651.

21.

Alt

Bitschnau

Osterling

, et al. The effects of combined gentamicin–hydroxyapatite coating for cementless joint prostheses on the reduction of infection rates in a rabbit infection prophylaxis model. Biomaterials 2006; 27(26): 4627–4634.

22.

Donath

Breuner

. A method for the study of undecalcified bones and teeth with attached soft tissues. The Säge-Schliff (sawing and grinding) techniquefied bones and teeth with attached soft tissues. The Sage–Schliff (sawing and grinding) technique. J Oral Pathol 1982; 11(4): 318–326.

23.

Isiklar

Darouiche

Landon

, et al. Efficacy of Antibiotics Alone for Orthopaedic Device Related Infections. Clin Orthop Relat Res 1996; 332: 184–189.

24.

Djanani

Mosheimer

Kaneider

, et al. Heparan sulfate proteoglycan-dependent neutrophil chemotaxis toward PR-39 cathelicidin. J Inflamm 2006; 3(3): 14.

25.

Post

Volk

, et al. PR39, a peptide regulator of angiogenesis. Nat Med 2000; 6: 49–55.

26.

Donlan

. Role of biofilms in antimicrobial resistance. ASAIO J 2000; 46(6): 47–52.

27.

Patel

. Biofilms and antimicrobial resistance. Clin Orthop Relat Res 2005; 437: 41–47.

28.

Mah

O'Toole

. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001; 9(1): 34–39.

29.

Cochran

McFeters

Stewart

. Reduced susceptibility of thin Pseudomonas aeruginosa biofilms to hydrogen peroxide and monochloramine. J Appl Microbiol 2000; 88: 22–30.

30.

Agerberth

Lee

Bergman

, et al. Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. Eur J Biochem 1991; 202(3): 849–854.

31.

Hennig-Pauka

Koch

Hoeltig

, et al. PR-39, a porcine host defence peptide, is prominent in mucosa and lymphatic tissue of the respiratory tract in healthy pigs and pigs infected with Actinobacillus pleuropneumoniae. BMC Res Notes 2012; 5: 539.

32.

Kumar

Kizhakkedathu

Straus

. Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules 2018; 8(1): 4.

33.

Linde

Hoffner

Refai

, et al. In vitro activity of PR-39, a proline-arginine-rich peptide, against susceptible and multi-drug-resistant Mycobacterium tuberculosis. J Antimicrob Chemother 2001; 47(5): 575–580.

34.

Zhou

. Apidaecin-type peptides: biodiversity, structure-function relationships and mode of action. Peptides 2006; 27(9): 2350–2359.

35.

Veldhuizen

EJ A

Schneider

VAF

Agustiandari

, et al. Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides. PLoS One 2014; 9(4): 95939.

36.

Mehra

Langkamer

Day

, et al. C reactive protein and skin temperature post total knee replacement. Knee 2005; 12(4): 297–300.

Protective effect of additional cathelicidin antimicrobial peptide PR-39 on prosthetic-joint infections

Abstract

Background

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Ethical statement

Cathelicidin lentivirus production

Isolation, cultivation and transduction of bone marrow stem cells

Expression of cathelicidin antimicrobial peptide PR-39 in bone marrow stem cells

PR-39 antimicrobial activity assays

Establish the PJI model of rabbit

Turnover of bone marrow stem cells in vivo

Histopathological examination

Statistical analysis

Results

Lentivirus vector were produced and transducted into bone marrow stem cells

Expression of PR-39 in bone marrow stem cells

Antimicrobial activity of PR-39 against Staphylococcus aureus in vitro

The protective effect of additional cathelicidin antimicrobial peptide PR-39 in orthopedic bacterial material infection

Osteogenic of BMSCs in vivo

Imaging results in vivo

Histological observation in vivo

Discussion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

ORCID iD

References