TITLE: Evaluation of the role of human β-defensin 3 in modulation of immunity and inflammatory response after knee replacement

  ABSTRACT:
The present study investigated the value of human β-defensin 3 (HBD-3) in adjusting the immunity and inflammatory response of T lymphocytes in the body after knee replacement. Sixty-four cases of knee replacement patients were successively selected and randomly divided into the control group and the observation group each with 32 cases. Once a day, for 7 days, patients in the control group were injected with placebo saline solution in the articular cavity. Levels of Th1 and/Th2, interleukin (IL)-2 and IL-10, tumor necrosis factor (TNF)-α, toll-like receptor (TLR)-4, and alkaline phosphatase (ALP) were compared one month later, and implant infection rates were compared within 1-year follow-up. Compared with patients in the control group, the levels of Th1 and Th1/Th2 in the observation group significantly increased, yet their Th2 decreased. The levels of IL-2 and TNF-α were also observed to be significantly elevated, yet IL-10 decreased. Furthermore, their TLR-4 and ALP levels were significantly higher. Three cases of implant-related infection occurred in the control group and 1 case in the observation group. In conclusion, HBD-3 could adjust the immunity and inflammatory response of cells in the body after knee replacement, possibly playing an important role in implant-related infection.

BODY.INTRODUCTION:
Total knee arthroplasty (TKA), is considered as one of the successful surgeries as it is able to eradicate knee joint pain effectively, recover knee joint function and improve the quality of life of the affected patients (1). Long-term study has proved that TKA provides a relatively higher survival rate (2), however, with a variety of complications, and their incidence rates vary (3). Complications mainly included formation of deep vein thrombosis and pulmonary embolism, prosthesis failure (including aseptic loosening, prosthesis wear or fracture, joint removal and liner out), nerve and vascular injury, formation of deep hematoma, periprosthetic fracture, knee stiffness, complications of patellofemoral (including patellofemoral instability, patella fracture, patellar clunk syndrome, extensor device fracture), pain and implant-related infection (4) which is the disastrous complication of TKA with incidence of 1–2.5% (5).  Staphylococcus aureus, is the main pathogen responsible for nosocomial infection, osteomyelitis and other infections related to orthopaedic implants (6). An earlier study showed that the bacteria transfer into osteocytes after infection could stimulate cytokine secreting, chemotactic factor and endogenous antimicrobial peptide β-defensin (7). This is a newly found bacterial infection inhibitor that is characterized by wide antimicrobial spectrum, rapid action, and a special action on immune regulation of bone infection (8). Based on in vitro cell and animal models, human β-defensin 3 (HBD-3) has the ability to regulate immunological activity of T, B lymphocyte, levels of interleukin (IL), tumor necrosis factor (TNF)-α, inflammatory factors, inhibit biofilm and is able to affect implant-related infection (9,10). The above applies to the TKA in clinic. Therefore, the present study explored the importance of HBD-3 in T lymphocyte response in the body after knee replacement.

BODY.PATIENTS AND METHODS.PATIENTS:
We selected 64 cases of patients who were admitted and underwent TKA treatment in Renmin Hospital of Wuhan University from January 2014 to June 2015, and the study obtained the approval of the Ethics Committee and written informed consent of the patient or their family. The patients were divided into the control group and the observation group each with 32 cases in accordance with the order of admission. Among which, 22 males and 10 females were in control group; aged from 38 to 76 years with an average age of 53.2±15.6 years; unilateral knee replacement was performed in 20 cases, bilateral knee replacement in 12 cases (5 cases in the same period and 7 cases in stages); primary disease: osseous arthritis in 16 cases, rheumatoid arthritis in 7 cases, traumatic arthritis in 6 cases and joint deformity in 3 cases; score of HSS before surgery was 32–65 with an average of 48.7±12.3 points; knee range of motion was 65–82° with an average of 76.7±8.3°. There were 20 males and 12 females in the observation group; aged from 41 to 78 years with an average age of 52.8±14.9 years; unilateral knee replacement was performed in 18 cases, bilateral knee replacement in 14 cases (5 cases in the same period and 9 cases in stages). Primary disease: osseous arthritis in 6 cases, rheumatoid arthritis in 6 cases, traumatic arthritis in 2 cases and joint deformity in 3 cases; score of HSS before surgery was 35–69 with an average of 51.3±15.6 points; knee range of motion was 63–85° with an average of 764.8±8.5°. The baseline data of two groups were comparable.

BODY.PATIENTS AND METHODS.RESEARCH METHOD:
The study of two groups was completed by the same surgery and nursing team in line with the standard medical procedure. The main steps of TKA: Notch at anterior median of knee joint, open up joint capsule to expose joint cavity, cut-off cruciate ligaments, remove joint marginal osteophyte and residual meniscus, release medial and lateral accessory ligament properly. In severe inversion deformity, attention was paid to the release of soft tissues around medial tibial plateau. Under the state of extreme genuflex, osteotomy of femur and tibia were carried out, osteotomy of 6° valgus and 3° external rotation at femoral condyle, of 5° backward at tibial plateau. Patella is not replaced routinely, so when repairing patella cartilage, an electric knife was used to scorch along the patella to block innervation and reduce the occurrence of anterior pain after surgery. The cemented prosthesis was placed when patellar tracking, stability of knee joint and ligament balance state were satisfactory. After surgery, 0° joint extension with brace protection was carried out.  The control group was given placebo saline solution of 10 ml in articular cavity, and the observation group HBD-3 (standard, 10 μg:50 μg:1 mg, Art no., CSB-P12186-3; Beijing Biodee Biotechnology Co., Ltd., Beijing, China) of 10 ml once a day, for 7 days.  To prevented the occurrence of deep vein thrombosis and pulmonary embolism, heparin sodium of 3,000 μ for subcutaneous injection was utilized for 1 week. Attention was paid to the observation of drainage, functional exercise of flexion and extension of quadriceps and knee joint were carried out. After removing the drainage tube, patients could walk with the help of the walking aid. The main method of functional exercise was to straighten knee joint actively. Intravenous analgesia pump was utilized to relieve pain and strive to achieve the state of extreme extension within 2 h with 90° flexion degree to the maximum.

BODY.PATIENTS AND METHODS.OBSERVATION INDICATORS:
Follow-up was until January 2016 and the average follow-up time was of 13 months. Comparison of Th1/Th2, IL-2 and IL-10, TNF-α, toll-like receptor (TLR)-4, alkaline phosphatase (ALP) levels and implant infection rate within 1-year follow-up was carried out. Th1/Th2 was detected by flow cytometry, IL-2, IL-10, TNF-α and TLR-4 levels were detected by ELISA method, and ALP level was detected by 2-amino-2-methyl-1-propanol (AMP), the ELISA kits of IL-2, IL-10, TNF-α and TLR-4 were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China) and experimental procedures were conducted according to the kit instructions.

BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS:
SPSS 20.0 software (IBM, Armonk, NY, USA) was used for statistical analysis, data were expressed by the mean ± standard deviation, comparisons among group were tested by Student's t-test, countable data were expressed as the number of case or a percentage, comparison within groups was tested by χ2 test, P<0.05 was considered to indicate a statistically significant difference.

BODY.RESULTS.COMPARISON OF TH1/TH2 LEVELS:
Th1 and Th1/Th2 of observation group was increased significantly comparing with that of control group, while Th2 decreased, the difference was statistically significant (P<0.05) (Table I and Fig. 1).

BODY.RESULTS.COMPARISON OF IL-2, IL-10 AND TNF-Α LEVELS:
IL-2 and TNF-α levels increased significantly comparing with that of control group, while IL-10 level decreased, the differences were statistically significant (P<0.05) (Table II).

BODY.RESULTS.COMPARISON OF TLR-4 AND ALP LEVELS:
TLR-4 and ALP levels of observation group were significantly higher than that of control group, the difference was statistically significant (P<0.05) (Table III).

BODY.RESULTS.COMPARISON OF IMPLANT INFECTION DURING THE FOLLOW-UP:
There were 3 cases of implant infection in control group (9.4%), 1 case in observation group (3.1%), there was no statistical significance (P=0.306, unilateral) by Fisher's exact probability method.

BODY.DISCUSSION:
Thorough debrigement and two-stage revision are currently recognized as gold standards for late infection treatment after TKA surgery (11). Spacers to hold thoroughly removed infected tissues are divided as static and dynamic, the two have similar effects in removing infection. However, the dynamic one retains bone mass and has relatively flexible knee joint, so that it can prevent soft tissue contracture and patella baja and help later prosthesis implantation and rapid preoperative recovery (12). In TKA failure cases, the daily habits of patients also count, and preoperative blood glucose control, cardiopulmonary function improvement, stopping smoking, weight control and lymphedema treatment can reduce the occurrence of infection (13).  Studies found that cells of infected parts could directly secrete β-defensin to resist bacteria and infection. β-defensin also participated in the regulation of innate immunity and adaptive immunity in the hosts (14). HBD-3 could also reduce CD98 expression and inhibited bacteria infection via competitively combining with surface receptor CD98 of the epithelial A549 cells (15). HBD-3 also regulated epithelial cells and fibroblasts to inhibit inflammation (16). Warnke et al found that under chronic inflammation in the body, osteoblast secretion of HBD-3 obviously increased and perhaps activated the immune system in bone tissues (17). Varoga et al found that Staphylococcus aureus supernatant could induce the release and expression of HBD-3 via TLR-2 and −4 on the surface of osteoblasts (18). So, endogenous antimicrobial peptide HBD-3 constituted the first line of defense after bacteria invaded the bone tissues.  The present study observed significant increases of Th1, Th1/Th2, IL-2 and TNF-α, decreases of Th2, IL-10, and obviously higher levels of TLR-4 and ALP, in the observation group as compared to control. Th1 cells mainly secreted IL-2, IFN-γ and TNF-α, mainly mediated immunity response relevant to cellular poison and local inflammation, assisted generation of antibodies and participated in cell immunity and delayed hypersensitivity inflammation. Th1 cells played an important role in intracellular pathogen infection of the body. Th2 cells mainly secreted IL-4, IL-5, IL-6, and IL-10, which mainly worked to stimulate the proliferation of B cells and generation of immunoglobulin G and antibody E and showed relation with immunity of body fluids. It has been proven that implant-related infection could induce the increase of Th1 cells and led to increase of Th1/Th2 (19). HBD-3 may regulate Th1/Th2, further enhanced the immunity response of cells and removed infection. The regulation process of Th1/Th2 by HBD-3 was related to increasing the expression of TLR-4 (20). ALP, secreted by osteoblasts, participated in the formation of new bone at the interface between implant and bone tissue. Three cases of implant-related infection occurred in the control group, and one such case in the observation group. No significant difference was detected due to the small sample size. However, it could be observed that HBD-3 regulated the immunity and inflammation of body cells after knee replacement, possibly playing an important role in implant-related infection. Further research need to be conducted in future on large sample sizes for more confirmatory results in clinical settings.

Question: Compared to injected with placebo saline solution in the articular cavity what was the result of HBD-3 (standard, 10 μg:50 μg:1 mg, Art no., CSB-P12186-3; Beijing Biodee Biotechnology Co., Ltd., Beijing, China) of 10 ml once a day, for 7 days. on TLR-4 and ALP levels?
Evidence: Th1 and Th1/Th2 of observation group was increased significantly comparing with that of control group, while Th2 decreased, the difference was statistically significant (P<0.05) (Table I and Fig. 1).
Answer: significantly increased