Electrical stimulation accelerates Wallerian degeneration and promotes nerve regeneration after sciatic nerve injury.

Following peripheral nerve injury (PNI), Wallerian degeneration (WD) in the distal stump can generate a microenvironment favorable for nerve regeneration. Brief low-frequency electrical stimulation (ES) is an effective treatment for PNI, but the mechanism underlying its effect on WD remains unclear. Therefore, we hypothesized that ES could enhance nerve regeneration by accelerating WD. To verify this hypothesis, we used a rat model of sciatic nerve transection and provided ES at the distal stump of the injured nerve. The injured nerve was then evaluated after 1, 4, 7, 14 and 21 days post injury (dpi). The results showed that ES significantly promoted the degeneration and clearance of axons and myelin, and the dedifferentiation of Schwann cells. It upregulated the expression of BDNF and NGF and increased the number of monocytes and macrophages. Through transcriptome sequencing, we systematically investigated the effect of ES on the molecular processes involved in WD at 4 dpi. Evaluation of nerves bridged using silicone tubing after transection showed that ES accelerated early axonal and vascular regeneration while delaying gastrocnemius atrophy. These results demonstrate that ES promotes nerve regeneration by accelerating WD and upregulating the expression of neurotrophic factors.

Biological characteristics of tissue engineered-nerve grafts enhancing peripheral nerve regeneration.

BACKGROUND: A favorable regenerative microenvironment is essential for peripheral nerve regeneration. Neural tissue-specific extracellular matrix (ECM) is a natural material that helps direct cell behavior and promote axon regeneration. Both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs) transplantation are effective in repairing peripheral nerve injury (PNI). However, there is no study that characterizes the in vivo microenvironmental characteristics of these two MSCs for the early repair of PNI when combined with neural tissue-derived ECM materials, i.e., acellular nerve allograft (ANA). METHODS: In order to investigate biological characteristics, molecular mechanisms of early stage, and effectiveness of ADSCs- or BMSCs-injected into ANA for repairing PNI in vivo, a rat 10 mm long sciatic nerve defect model was used. We isolated primary BMSCs and ADSCs from bone marrow and adipose tissue, respectively. First, to investigate the in vivo response characteristics and underlying molecular mechanisms of ANA combined with BMSCs or ADSCs, eighty-four rats were randomly divided into three groups: ANA group, ANA+BMSC group, and ANA+ADSC group. We performed flow cytometry, RT-PCR, and immunofluorescence staining up to 4 weeks postoperatively. To further elucidate the underlying molecular mechanisms, changes in long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) were systematically investigated using whole transcriptome sequencing. We then constructed protein-protein interaction networks to find 10 top ranked hub genes among differentially expressed mRNAs. Second, in order to explore the effectiveness of BMSCs and ADSCs on neural tissue-derived ECM materials for repairing PNI, sixty-eight rats were randomized into four groups: ANA group, ANA+BMSC group, ANA+ADSC group, and AUTO group. In the ANA+BMSC and ANA+ADSC groups, ADSCs/BMSCs were equally injected along the long axis of the 10-mm ANA. Then, we performed histological and functional assessments up to 12 weeks postoperatively. RESULTS: The results of flow cytometry and RT-PCR showed that ANA combined with BMSCs exhibited more significant immunomodulatory effects, as evidenced by the up-regulation of interleukin (IL)-10, down-regulation of IL-1ß and tumor necrosis factor-alpha (TNF-α) expression, promotion of M1-type macrophage polarization to M2-type, and a significant increase in the number of regulatory T cells (Tregs). ANA combined with ADSCs exhibited more pronounced features of pro-myelination and angiogenesis, as evidenced by the up-regulation of myelin-associated protein gene (MBP and MPZ) and angiogenesis-related factors (TGF-ß, VEGF). Moreover, differentially expressed genes from whole transcriptome sequencing results further indicated that ANA loaded with BMSCs exhibited notable immunomodulatory effects and ANA loaded with ADSCs was more associated with angiogenesis, axonal growth, and myelin formation. Notably, ANA infused with BMSCs or ADSCs enhanced peripheral nerve regeneration and motor function recovery with no statistically significant differences. CONCLUSIONS: This study revealed that both ANA combined with BMSCs and ADSCs enhance peripheral nerve regeneration and motor function recovery, but their biological characteristics (mainly including immunomodulatory effects, pro-vascular regenerative effects, and pro-myelin regenerative effects) and underlying molecular mechanisms in the process of repairing PNI in vivo are different, providing new insights into MSC therapy for peripheral nerve injury and its clinical translation.

Immunomodulatory effects of mesenchymal stem cells in peripheral nerve injury.

Various immune cells and cytokines are present in the aftermath of peripheral nerve injuries (PNI), and coordination of the local inflammatory response is of great significance for the recovery of PNI. Mesenchymal stem cells (MSCs) exhibit immunosuppressive and anti-inflammatory abilities which can accelerate tissue regeneration and attenuate inflammation, but the role of MSCs in the regulation of the local inflammatory microenvironment after PNI has not been widely studied. Here, we summarize the known interactions between MSCs, immune cells, and inflammatory cytokines following PNI with a focus on the immunosuppressive role of MSCs. We also discuss the immunomodulatory potential of MSC-derived extracellular vesicles as a new cell-free treatment for PNI.

[Clinical efficacy of different rehabilitation modes for lumbar degenerative diseases after operation].

OBJECTIVE: To compare clinical effects of different postoperative rehabilitation modes on lumbar degenerative diseases, and explore influence of rehabilitation mode and other factors on postoperative effect. METHODS: From June 2013 to July 2016, totally 900 patients were admitted from nine tertiary hospitals in Beijing to perform single segment bone grafting and internal fixation due to lumbar degenerative diseases were prospectively analyzed. There were 428 males and 472 females, the age of patient over 18 years old, with an average of (51.42±12.41) years old;according to patients' subjective wishes and actual residence conditions, all patients were divided into three groups, named as observation group 1 (performed integrated rehabilitation approach and orthopedic treatment model intervention), observation group 2 (performed integrated rehabilitation approach and orthopedic treatment, classified rehabilitation model intervention), and control group(performed routine rehabilitation model intervention). Visual analogue scale(VAS), Oswestry Disability Index(ODI) and Japanese Orthopaedic Association (JOA) were used to evaluate postoperative efficacy among three groups at 24 weeks. Possible factors affecting the postoperative efficacy including age, age grouping, gender, body mass index (BMI), BMI grouping, education level, visiting hospital, payment method of medical expenses, preoperative complications, preoperative JOA score, clinical diagnosis, surgery section, operative method, intraoperative bleeding volume, postoperative complications and rehabilitation mode were listed as independent variables, and postoperative ODI score at 24 weeks as dependent variables. Univariate analysis was used to analyze relationship between influencing factors and postoperative efficacy. Multiple linear regression was used to analyze relationship between influencing factors, rehabilitation mode and postoperative ODI score at 24 weeks, in further to find out the main reasons which affect postoperative efficacy, and to analyze impact of rehabilitation mode on postoperative efficacy. RESULTS: All patients were followed up for 24 weeks after operation. All incisions healed at stage I with stable internal fixation. (1)Evaluation of postoperative efficacy:① There were no statistical differences in preoperative VAS and ODI among three groups(P>0.05), the degree of pain and dysfunction decreased among three groups after operation, and had differences in postoperative VAS and ODI among three groups (P<0.05). There were no significant differences between observation group 1 and observation group 2(P>0.05); while compared with observation group 1 and control group, observation group 2 and control group, there were significant differences (P<0.05). ②The function among three groups were improved in varying degrees after operation. There was difference in JOA score among three groups before operation and 24 weeks after operation (P<0.05). There were no difference in JOA score among three groups between observation group 1 and observation group 2 (P>0.05);while compared with observation group 1 and control group, observation group 2 and control group, there were significant differences (P<0.05). (2)Influencing factors at 24 weeks after operation:①Univariate analysis showed gender, age, age grouping, education level, preoperative complications, clinical diagnosis, operative section, operative method, preoperative JOA score and rehabilitation mode had statistical significance with postoperative ODI score at 24 weeks (P<0.05). BMI, BMI grouping, payment method of medical expenses, visiting hospital, intraoperative bleeding volume, postoperative complications had no statistical significance with postoperative ODI score at 24 weeks (P<0.05).②Multivariate analysis results showed gender, rehabilitation mode, age, preoperative JOA score entered the equation eventually, stepwise multiple linear equation obtained had statistical significance (F=12.294, P= 0.000). Among rehabilitation mode, standardized regression coefficient of the integrated rehabilitation approach and orthopedic treatment with classified rehabilitation model was absolute value of the largest (0.176), which had the greatest influence on postoperative curative effect. The degree of dysfunction in control group was higher than that in observation group 1 and observation group 2. Postoperative dysfunction was more severe in males than that of in females. Older age has higher degree of dysfunction after operation. Lower preoperative JOA score has higher degree of dysfunction after operation. CONCLUSION: Preoperative JOA score, gender, age could predict postoperative clinical effects of lumbar degenerative diseases in varying degrees treated with single level bone graft fusion and internal fixation. Different rehabilitation modes could improve clinical effects. Intergrated rehabilitation orthopedic treatment model and integrated rehabilitation approach and orthopedic treatment with classifiedrehabilitation model are superior to conventional rehabilitation model in improving patients' postoperative function and relieving pain, which is worthy of promoting in clinical.

Neuroprotective effects of curcumin alleviate lumbar intervertebral disc degeneration through regulating the expression of iNOS, COX­2, TGF­ß1/2, MMP­9 and BDNF in a rat model.

Curcumin is a natural product with antimutagenic, antitumor, antioxidant and neuroprotective properties. However, to the best of our knowledge, curcumin has yet to be investigated for the treatment of lumbar intervertebral disc degeneration LIDD). The aim of the present study was to investigate whether curcumin can alleviate LIDD through regulating the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)­2, transforming growth factor (TGF)­ß1/2, matrix metalloproteinase (MMP)­9 and brain­derived neurotrophic factor (BDNF) in a rat model of LIDD. The results of the present study suggest that pretreatment with curcumin can prevent the development of LIDD in rats. It was revealed that treatment with curcumin significantly reduced interleukin (IL)­1ß and IL­6, iNOS, COX­2 and MMP­9 levels in rats with LIDD. In addition, treatment with curcumin reduced the mRNA expression levels of TGF­ß1 and TGF­ß2, whereas it increased the mRNA expression levels of BDNF in rats with LIDD. In conclusion, the present findings indicate that curcumin may exert protective effects on LIDD development, exerting its action through the regulation of iNOS, COX­2, TGF­ß1/2, MMP­9 and BDNF.

[Influences of decellularization processes on immunogenicity of chemically acellular nerve allografts].

OBJECTIVE: To investigate the relationship between immunogenicity and decellularization processes of chemically acellular nerve allografts. METHODS: Adult Sprague Dawley rats were used as nerve donors and adult male Wistar rats used as nerve recipient hosts. 25 mm nerve segments were excised from SD rats' sciatic nerves. The nerve segments were decellularized via an improved chemical decelluarization treatment as follows: (1) nerve segments were rinsed with cold sterile Ringer's solution; (2) stabilized by pinning the ends to a thin plastic support, and submerged in 4% Triton-100 solution 12 h; (3) soaked into 3% sodium deoxycholate for 12 h; (4) washed in distilled water for 6 h. The procedures were repeated once again. The acellular nerve allografts from SD rats were sterilized by gamma irradiation and implanted into Wistar rats subcutanously. The control group was implantation of fresh nerve allografts from SD rats. The immunogenicity of acellular nerve allograft was tested by immunohistochemical examination of the intensity of CD3(+), CD4(+) and CD8(+) cells that infiltrated the allografts. Ulnar nerve segments were obtained from forearms of dogs and decellularized according to above procedures. According as the decellularization times, The ulnar nerve segments were divided into three subgroups: in group I, group II and group III, the nerve segments were decellularized repeatedly two, three and four cycles respectively. Each ulnar nerve segment was subdivided into five portions from proximal to distal end. The degrees of decellularization, demyelination and basal lamina integrity of extracellular matrix scaffold were observed with microscope and assessed by a score system. The immunohistochemical staining of GAG was observed. RESULTS: The intensity of CD3(+), CD4(+) and CD8(+) T cells that infiltrated the allografts was greatly lower in acellular nerves than in fresh nerves. The mild cell-mediated host-graft immunorejection in acellular nerves was observed. On the decellularization procedures, the cells were completely extracted from nerves in all groups, but the myelin sheath were partially existed, and the GAG was present in the basal membrane of myelin sheath. In the score of demyelination, there were no statistical differences between groups (P > 0.05). The statistical difference of basal lamina integrity scores between group I and group II, group I and group III were significant (P < 0.05). As increasing the times of process, the degrees of disintegrity of basal lamina was significantly enhanced. CONCLUSIONS: Although decellularization processes significantly reduce the cell-mediated immunorejection of acellular nerve allografts, it can induce mild immunoreaction all the same, the antigen that responsible for immunogenicity may be the residual component of GAG in myelin sheath.

[The clinical application and biomechanical study of reconstruction of anterior and posterior cruciate ligament with hamstring tendons knot implant fixation].

OBJECTIVE: To investigate the feasibility of reconstruction of anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) with hamstring tendons knot implant fixation. METHODS: Fifty-two cases of ACL and PCL old injury were reconstructed under the arthroscopy with double bundles hamstring tendons knot implant fixated in the bottle-necked femoral tunnel. The tibia side were fixated by tendon weave suture cross tied a knot at the bone bridge of tibia. In this group reconstruction of ACL was in 25, PCL and ACL at the same time reconstruction in 15, PCL in 12. The failure test and displacement evaluation were used to study the biomechanics of reconstruction of ACL by hamstring tendon knot implant fixation (n = 13) in porcine knees. The control group were reconstructed with bone-patellar tendon-bone B-PT-B and interference screw (n = 11). The tibia side tendon weave suture immobility by cross tied a knot at the tibia bone bridge (n = 7) and interference screw in the tibia tunnel (n = 8). RESULTS: Forty-nine cases were followed up, average 14.6 months. The results of Lanchman test was negative in 46 cases, positive in 3 cases. Preoperative Lysholm score was 56.7, and postoperative was 92.8. According to the knee joint effective evaluate standard, 46 were excellent and 3 good. The maximal pull-out force of hamstring tendons knot implanted fixation had been greater than B-PT-B fixation by interference screw. There were similar displacement in 100 N and 400 N load between hamstring tendons knot implant fixation and B-PT-B (P > 0.05). Peak force test maximal displacement evaluation and failure energy absorption measurement showed that hamstring tendons knot implant fixation was significantly greater than B-PT-B (P < 0.01). The failure test and displacement evaluation of tendon fixation by weave suture knot at the tibia bone bridge cross tie were greater than B-PT-B fixation by interference screw. CONCLUSION: The methods of reconstruct of ACL and PCL by hamstring tendons knot implant fixation are feasible. The advantages include: it is biological fixation, not machine fixation; it may benefit for tendons with bone tunnel healing and decrease medical cost. Decreasing or eliminating displacement is helpful to prevent relaxation after reconstruction of ACL and PCL.

Treatment and Rehabilitation of Knee Joints Straight Stiffness After Burns.

The knee release surgery and postoperative rehabilitation of patients after burns and knee straight stiffness were investigated. Eleven patients were treated for 16 side burns and knee stiffness who consisted of nine males and two females, aged 19 to 54 years (mean = 33.2). The duration of the patients' knee stiffness ranged from 8 to 26 months, with an average of 12.6 months. Their preoperative flexion ranged from 5° to 50°, with an average of 26.2°. Their preoperative Hospital for Special Surgery (HSS) knee scores ranged from 46 to 72 points, with an average of 55.8 points. All stiff knees were treated with release surgery, along with total release of intra-articular adhesion and excision of vastus intermedius. After the arthrolysis of the stiff knee joint, the tight skin was completely loose in the adhesions. The soft tissue contracture was not grafted, but the shade fascia was freed to increase skin ductility. All knee joints were released to more than 90° of flexion in the operation, and reversed fascia flaps were used to suture the loss of the deep fascia at the position of flexion of 90°. After the operation, the knee joint was fixed in flexion for 72 h while being actively cared for by early rehabilitation. Subsequently, the patient's skin coverage, joint motion, and joint function recovery were observed. Based on the follow-up of the patients for the following 16 to 36 months (mean = 25.7), the knee flexion of the patients ranged from 110° to 135°, with an average of 122.2° and 96° increase (P < 0.01). Furthermore, the patients had better skin ductility to meet the increase in joint flexion. HSS knee function scores at the end of follow-up ranged from 93 to 100 points, with an average of 97.5 points and an increase of 41.7 points (P < 0.01). The joint function improved significantly. The arthrolysis of straight stiff knee joints after burns can ease muscle contracture and free the shade fascia, thus avoiding the need to undergo skin grafting. Short-term fixation of the joints after surgery and active flexion rehabilitation may also be the key to improve skin ductility and joint function of the patients.

[Effects of acupuncture intervention at different stages on urinary function reconstruction of neurogenic bladder after spinal cord injury].

OBJECTIVE: To explore the effects of early intervention of acupuncture on urinary function reconstruction of neurogenic bladder after spinal cord injury, so as to find the optimal treatment timing of acupuncture. METHODS: Fifty-three patients of neurogenic bladder after spinal cord injury were randomly divided into a prior-to-catheter group (25 cases) and a posterior-to-catheter group (28 cases). For the prior-to-catheter group, one week before the removal of catheter, acupoints on the abdomen and back such as Guanyuan (CV 4), Zhongji (CV 3), Baliao (Shangliao (BL 31), Ciliao (BL 32), Zhongliao (BL 33), Xialiao (BL 34), Jiaji (EX-B 2) and acupoints at distal end such as Zusanli (ST 36) and Sanyinjiao (SP 6) were selected; after the catheter removal, simple method. of water column was used to measure bladder capacity pressure to classify the type of bladder, and then different acupuncture manipulations were given; intermittent catheterization was performed if condition was allowed. The posterior-to-catheter group applied identical treatment after removal of catheter as the prior-to-catheter group. The treatment was given 5 times per week; after one-month treatment, five aspects including the time of first effective urination, time of establishing reflex urination, average residual volume of urine, time of residual urine less than 100 mL and quality of life (QOL) were evaluated. RESULTS: After treatment, the time of first effective urination, time of establishing reflex urination, average residual volume of urine and time of residual urineless than 100 mL in the prior-to-catheter group were all superior to those in the posterior-to-catheter group (all P<0. 05) ; the score of QOL was improved in two groups after treatment (both P<0. 01), but the difference between two groups was not statistically significant (P>0. 05); each index of urination function was not significant among patients with different injured segments of spinal cord (all P>0. 05). CONCLUSION: The early intervention of acupuncture (prior to catheter) has obvious improving function on establishing balanced bladder in patients with neurogenic bladder after spinal cord injury, which is superior to acupuncture intervention after removal of catheter, however, the effects of different injured segments on establishing balanced bladder are not different.

Hypoxia-Inducible Factor 1-Alpha Regulates Cancer-Inhibitory Effect of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been shown to be able to inhibit cancer cells growth. In this study, we investigate the role and the molecular mechanism of hypoxia-inducible factor 1-alpha (HIF-1α) in inhibition of cancer cell proliferation by human MSCs through depletion and overexpression of HIF-1α in human MSCs. We found that the cell culture medium from HIF-1α-depleted Z3 cells significantly promotes breast cancer MCF-7 cell proliferation and colony formation. The expression of p21 is increased in MCF-7 cells, but p53 level remains unchanged. In contrast, the cultured medium from HIF-1α-overexpressed Z3 cells dramatically inhibits MCF-7 cell proliferation and colony formation. The expression of p21 is inhibited in MCF-7 cells, but p53 does not change. We conclude HIF-1α promotes inhibitory effect of human MCSs on breast cancer cell proliferation and colony formation. This process is tightly correlated with cell cycle protein p21 level in cancer cells.