Extracellular vesicles produced by 3D cultured MSCs promote wound healing by regulating macrophage activation through ANXA1.

The conundrum of wound healing has transformed into an imminent medical challenge. Presently, cell-free therapy centered around extracellular vesicles (EVs) has become a pivotal and promising research avenue. EVs generated from three-dimensional (3D) cell cultures have been previously established to possess enhanced tissue regeneration potential, although the underlying mechanisms remain elusive. In this study, we observed higher expression of annexin ANXA1 in 3D-cultured EVs. Remarkably, 3D-EVs with elevated ANXA1 expression demonstrated a more potent capacity to promote macrophage polarization from the M1 phenotype to the M2 phenotype. Concurrently, they exhibited superior abilities to enhance cell migration and tube formation, facilitating expedited wound healing in animal experiments. Conversely, the application of an ANXA1 inhibitor counteracted the positive effects of 3D-EVs. Taken together, our data validate that extracellular vesicles derived from 3D-cultured MSCs regulate macrophage polarization via ANXA1, thereby fostering wound healing.

Comparison between Novel Anatomical Locking Guide Plate and Conventional Locking Plate for Acetabular Fractures: A Finite Element Analysis.

The treatment of complex acetabular fractures remains a complicated clinical challenge. Our self-designed novel anatomical locking guide plate (NALGP) has previously shown promising potential in T-shaped acetabular fractures (TAF), but a direct comparison with conventional fixations is yet to be made. The TAF model was established based on a volunteer's computer tomography data and then fixed with double column locking plates (DLP), a posterior column locking plate with anterior column screws (LPACS), and our NALGP. Forces of 200 N, 400 N, and 600 N were then loaded on the model vertically downward, respectively. The stress distribution and peaks and maximum displacements at three sites were assessed. We found that the stress area of all three plates was mainly concentrated around the fracture line, while only the matching screws of the NALGP showed no obvious stress concentration points. In addition, the NALGP and DLP showed significantly less fracture fragment displacement than the LPACS at the three main fracture sites. The NALGP was found to have less displacement than DLP at the posterior column and ischiopubic branch sites, especially under the higher loading forces of 400 N and 600 N. The fixation stability of the NALGP for TAF was similar to that of DLP but better than that of LPACS. Moreover, the NALGP and its matching screws have a more reasonable stress distribution under different loads of force and the same strength as the LPACS.

The dual role of microglia in neuropathic pain after spinal cord injury: Detrimental and protective effects.

Spinal cord injury (SCI) is a debilitating condition that is frequently accompanied by neuropathic pain, resulting in significant physical and psychological harm to a vast number of individuals globally. Despite the high prevalence of neuropathic pain following SCI, the precise underlying mechanism remains incompletely understood. Microglia are a type of innate immune cell that are present in the central nervous system (CNS). They have been observed to have a significant impact on neuropathic pain following SCI. This article presents a comprehensive overview of recent advances in understanding the role of microglia in the development of neuropathic pain following SCI. Specifically, the article delves into the detrimental and protective effects of microglia on neuropathic pain following SCI, as well as the mechanisms underlying their interconversion. Furthermore, the article provides a thorough overview of potential avenues for future research in this area.

Research Progress in Abrasive Water Jet Processing Technology.

Abrasive water jet machining technology is an unconventional special process technology; its jet stream has high energy, and its machining process is characterized by no thermal deformation, no pollution, high applicability, and high flexibility. It has been widely used for processing different types of materials in different fields. This review elaborates on the basic principles and characteristics of abrasive water jet processing, the mechanism of erosion, the simulation of the processing, the influence of process parameters in machining removal, and the optimization of improvements, as well as introduces the current application status, new technology, and future development direction of abrasive water jet technology. This review can provide an important information reference for researchers studying the machining processing of abrasive water jet technology.

Early-phase rotator training impairs tissue repair and functional recovery after spinal cord injury.

Spinal cord injury (SCI) is a devastating disorder that often results in severe sensorimotor function impairment with limited recovery of function. In recent years, rehabilitation training for spinal cord injury has gradually emerged, and some of them play an important role in the repair of spinal cord injury However, the optimal training regimen for SCI remains to be determined. In this study, we explore the effects of rotarod training (began at 7 days post-injury) on the recovery of motor function after SCI, as well as its possible repair mechanism from the aspects of function and histopathological changes, the behaviors of specific trophic factors and cytokines, and the expression profile of specific genes. Multiple functional assessments showed that rotarod training initiated at 7 days post-injury is unsuitable for promoting neuro-electrophysiological improvement and trunk stability, but impaired functional coordination and motor recovery. In addition, rotarod training has negative effects on spinal cord repair after SCI, which is manifested as an increase of lesion area, a decrease in neuronal viability, a deterioration in immuno-microenvironment and remyelination, a significant reduction in the expression of trophic factors and an increase in the expression of pro-inflammatory factors. RNA sequencing suggested that the genes associated with angiogenesis and synaptogenesis were significantly downregulated and the PI3K-AKT pathway was inhibited, which was detrimental to spinal cord repair and impeded nerve regeneration. These results indicate that immediate rotarod training after SCI is currently unsuitable for rehabilitation in mice.

Application of photocrosslinkable hydrogels based on photolithography 3D bioprinting technology in bone tissue engineering.

Bone tissue engineering (BTE) has been proven to be an effective method for the treatment of bone defects caused by different musculoskeletal disorders. Photocrosslinkable hydrogels (PCHs) with good biocompatibility and biodegradability can significantly promote the migration, proliferation and differentiation of cells and have been widely used in BTE. Moreover, photolithography 3D bioprinting technology can notably help PCHs-based scaffolds possess a biomimetic structure of natural bone, meeting the structural requirements of bone regeneration. Nanomaterials, cells, drugs and cytokines added into bioinks can enable different functionalization strategies for scaffolds to achieve the desired properties required for BTE. In this review, we demonstrate a brief introduction of the advantages of PCHs and photolithography-based 3D bioprinting technology and summarize their applications in BTE. Finally, the challenges and potential future approaches for bone defects are outlined.

P53 negatively regulates the osteogenic differentiation in jaw bone marrow MSCs derived from diabetic osteoporosis.

Patients with diabetic osteoporosis (DOP) often suffer from poor osseointegration of artificial implants, which is a challenge that affects implant outcomes. The osteogenic differentiation ability of human jaw bone marrow mesenchymal stem cells (JBMMSCs) is the key to implant osseointegration. Studies have shown that the microenvironment of hyperglycemia affects the osteogenic differentiation of mesenchymal stem cells (MSC), but the mechanism is still unclear. Therefore, the aim of this study was to isolate and culture JBMMSCs from surgically derived bone fragments from DOP patients and control patients to investigate the differences in their osteogenic differentiation ability and to elucidate its mechanisms. The results showed that the osteogenic ability of hJBMMSCs was significantly decreased in the DOP environment. Mechanism study showed that the expression of senescence marker gene P53 was significantly increased in DOP hJBMMSCs compared to control hJBMMSCs according to RNA-sequencing result. Further, DOP hJBMMSCs were found to display significant senescence using ß-galactosidase staining, mitochondrial membrane potential and ROS assay, qRT-PCR and WB analysis. Overexpression of P53 in hJBMMSCs, knockdown of P53 in DOP hJBMMSCs, and knockdown followed by overexpression of P53 significantly affected the osteogenic differentiation ability of hJBMMSCs. These results suggest that MSC senescence is an important reason for decreasing osteogenic capacity in DOP patients. P53 is a key target in regulating hJBMMSCs aging, and knocking down P53 can effectively restore the osteogenic differentiation ability of DOP hJBMMSCs and promote osteosynthesis in DOP dental implants. It provided a new idea to elucidate the pathogenesis and treatment of diabetic bone metabolic diseases.

Synchronized activity of sensory neurons initiates cortical synchrony in a model of neuropathic pain.

Increased low frequency cortical oscillations are observed in people with neuropathic pain, but the cause of such elevated cortical oscillations and their impact on pain development remain unclear. By imaging neuronal activity in a spared nerve injury (SNI) mouse model of neuropathic pain, we show that neurons in dorsal root ganglia (DRG) and somatosensory cortex (S1) exhibit synchronized activity after peripheral nerve injury. Notably, synchronized activity of DRG neurons occurs within hours after injury and 1-2 days before increased cortical oscillations. This DRG synchrony is initiated by axotomized neurons and mediated by local purinergic signaling at the site of nerve injury. We further show that synchronized DRG activity after SNI is responsible for increasing low frequency cortical oscillations and synaptic remodeling in S1, as well as for inducing animals' pain-like behaviors. In naive mice, enhancing the synchrony, not the level, of DRG neuronal activity causes synaptic changes in S1 and pain-like behaviors similar to SNI mice. Taken together, these results reveal the critical role of synchronized DRG neuronal activity in increasing cortical plasticity and oscillations in a neuropathic pain model. These findings also suggest the potential importance of detection and suppression of elevated cortical oscillations in neuropathic pain states.

A Novel Anatomical Locking Guide Plate for Treating Acetabular Transverse Posterior Wall Fracture: A Finite Element Analysis Study.

OBJECTIVE: To improve the treatment of the acetabular transverse posterior wall fracture (ATPWF), a novel anatomical locking guidance plate (NALGP) was designed and compared with traditional fixations using finite element analysis. METHODS: The ATPWF model was constructed using the three-dimensional finite element model of the half pelvis via the Mimics software and three internal devices were used to fix this model: the posterior-column locking plate with anterior-column screws (PCLP), double-column locking plates (DCLP), and NALGP. Next, mesh division was conducted by solid 187 tetrahedral elements in the workbench software. After defining the boundary condition and material properties, each assembly model was loaded in an increasing manner with a downward vertical force of 200, 400, and 600 N, respectively. The loading force was directed at 45 degrees upward in the coronal plane and 25 degrees backward in the sagittal plane. Finally, the stress distribution and stress peak of plates and screws were measured and evaluated, and the displacement of fracture fragments under different loading force was assessed among the three groups. RESULTS: For stress distribution, it was found that the stress mainly acted on the posterior-column plate, especially concentrated at the middle and lower section of the plate in all three groups after fixation on the ATPWF. In addition, most stresses of screws appeared on the lag screws instead of the common screws. The common screws in the NALGP group experienced larger stresses under all loading force, while those in the DCLP group withstood less stresses compared to those in the PCLP group. For the displacement of fracture fragments, the NALGP group were found to have less fracture fragment displacements than the PCLP group, but had comparable results to DCLP at both the transverse fracture and the posterior wall fracture sites. CONCLUSION: The newly-designed fixation device showed superiorities on fracture stabilization over PCLP, but had comparable stability to DCLP. This suggests that the DCLP might be unnecessary for treating ATPWF in some instances because it might cause bigger surgical trauma and blood loss.

Emerging Roles of Microglia Depletion in the Treatment of Spinal Cord Injury.

Microglia, as the resident immune cells and first responder to neurological insults, play an extremely important role in the pathophysiological process of spinal cord injury. On the one hand, microglia respond rapidly and gather around the lesion in the early stage of injury to exert a protective role, but with the continuous stimulation of the injury, the excessive activated microglia secrete a large number of harmful substances, aggravate the injury of spinal cord tissue, and affect functional recovery. The effects of microglia depletion on the repair of spinal cord injury remain unclear, and there is no uniformly accepted paradigm for the removal methods and timing of microglia depletion, but different microglia depletion strategies greatly affect the outcomes after spinal cord injury. Therefore, this review summarizes the physiological and pathological roles of microglia, especially the effects of microglia depletion on spinal cord injury-sustained microglial depletion would aggravate injury and impair functional recovery, while the short-term depletion of microglial population in diseased conditions seems to improve tissue repair and promote functional improvement after spinal cord injury. Furthermore, we discuss the advantages and disadvantages of major strategies and timing of microglia depletion to provide potential strategy for the treatment of spinal cord injury.

Establishment and characterization of an induced pluripotent stem cell line CPGHi004-A from peripheral blood mononuclear cells of a healthy adult.

Induced pluripotent stem cells (iPSCs) are regarded as ideal cell sources of neural stem cells for the treatment of spinal cord injury (SCI). To minimize the damages to donors, we sought to generate iPSC lines from peripheral blood mononuclear cells (PBMCs) of a healthy adult using non-integrated episomal reprogramming vectors. We confirmed that generated iPSCs had normal karyotype, expressed classical stemness surface markers, and managed to form teratoma with three typical germ layers in vivo. This cell line would be a valuable cell source for high-efficient generation of nonrenewable cells like neural stem cells for treating SCI.

3D spheroids of human placenta-derived mesenchymal stem cells attenuate spinal cord injury in mice.

Mesenchymal stem cell (MSC) is an absorbing candidate for cell therapy in treating spinal cord injury (SCI) due to its great potential for multiple cell differentiation, mighty paracrine secretion as well as vigorous immunomodulatory effect, of which are beneficial to the improvement of functional recovery post SCI. However, the therapeutic effects of MSC on SCI have been limited because of the gradual loss of MSC stemness in the process of expanding culture. Therefore, in this study, we aimed to maintain those beneficial properties of MSC via three-dimensional spheroid cell culture and then compared them with conventionally-cultured MSCs in the treatment of SCI both in vitro and in vivo with the aid of two-photon microscope. We found that 3D human placenta-derived MSCs (3D-HPMSCs) demonstrated a significant increase in secretion of anti-inflammatory factors and trophic factors like VEGF, PDGF, FGF via QPCR and Bio-Plex assays, and showed great potentials on angiogenesis and neurite morphogenesis when co-cultured with HUVECs or DRGs in vitro. After transplantation into the injured spinal cord, 3D-HPMSCs managed to survive for the entire experiment and retained their advantageous properties in secretion, and exhibited remarkable effects on neuroprotection by minimizing the lesion cavity, inhibiting the inflammation and astrogliosis, and promoting angiogenesis. Further investigation of axonal dieback via two-photon microscope indicated that 3D-HPMSCs could effectively alleviate axonal dieback post injury. Further, mice only treated with 3D-HPMSCs obtained substantial improvement of functional recovery on electrophysiology, BMS score, and Catwalk analysis. RNA sequencing suggested that the 3D-HPMSCs structure organization-related gene was significantly changed, which was likely to potentiate the angiogenesis and inflammation regulation after SCI. These results suggest that 3D-HPMSCs may hold great potential for the treatment of SCI.

Comparative study of the reorganization in bilateral motor and sensory cortices after spinal cord hemisection in mice.

OBJECTIVE: The effects of spinal cord injury (SCI) on sensorimotor cortex plasticity have not been well studied. Therefore, to explore the reorganization after SCI, we dynamically monitored postsynaptic dendritic spines of pyramidal neurons in vivo. METHODS: Thy1-YFP transgenic mice were randomly divided into two groups: the control and SCI group. We then opened the spinal vertebral plates of all mice and sectioned one-half of the spinal cord in SCI group. The relevant areas were imaged bilaterally at 0, 3, 14 and 28 days post-SCI. The rates of elimination, formation and stable spines were evaluated. RESULTS: At the early stage, the rate of stable and elimination spines experienced a similar change trend. But the rate of formation spines in the contralateral sensory cortex was significantly increased after SCI compared with those in the control group. At the late stage, spines of three types remodeled very differently between the sensory and motor cortex. Compared with those in the control group, spines in the bilateral sensory cortex demonstrated obvious differences in the rate of stable and elimination spines but not formation spines, while spines in the motor cortex, especially in the contralateral cortex increased significantly in the rate of formation after SCI. As for survival rate, differences mainly appeared in time frame instead of cortex type or region. CONCLUSIONS: The dendritic spines in hindlimb representation area of the sensorimotor cortex experienced bilaterally remodeling after SCI. And those spines in the sensory and motor cortex experienced great but different change trends after SCI.

A systematic review and meta-analysis comparing arthroplasty and internal fixation in the treatment of elderly displaced femoral neck fractures.

BACKGROUND: Currently, there are 2 mainstream treatments for displaced femoral neck fracture, including internal fixation and arthroplasty. However, there are still some controversial problems as to which treatment should be primarily chosen. METHODS: The relevant studies comparing arthroplasty with internal fixation were searched in the databases of PubMed, Embase, and Cochrane Library. Finally, 31 relevant randomized controlled trials were included in this meta-analysis. The quality of studies was evaluated and meta-analyses were performed using RevMan 5.3 software. We also assessed the heterogeneity among studies and publication bias via the I-squared index and forest plots. RESULTS: There was no significant difference between arthroplasty and internal fixation groups in patient mortality at both short-term and long-term points. However, patients treated with arthroplasty showed significantly lowered risks of reoperation both at short-term (5.6% vs 31.5%; relative risks (RR)  = 0.19; 95% CI, 0.13-0.28; P < .00001) and long-term follow-up (9.5% vs 45.9%; RR = 0.23; 95% CI, 0.17-0.33; P < .00001). Similarly, arthroplasty-treated patients demonstrated a significant decrease in the risk of postoperation complications at short-term (10.3% vs 34.4%; RR = 0.37, 95% CI, 0.24-0.57; P < .00001) and long-term follow-up (11.7% vs 42.5%; RR = 0.30, 95% CI, 0.16-0.57; P < .0002). Besides, patients in the arthroplasty group were associated with better alleviation of pain postoperation (18.3% vs 31.1%; RR = 0.50, 95% CI, 0.33-0.78; P = .002).In trial sequence analyses, all cumulative Z curves except that of mortality crossed the trial sequential monitoring boundaries and conventional boundaries, and required information size has been reached. CONCLUSIONS: Arthroplasty leads to a lower rate of reoperation, a reduced risk of complications, and a better alleviation of postoperation pain both at short-term and long-term follow-up. Most importantly, and according to trial sequence analyses, more than enough evidence has been achieved that arthroplasty does show better outcomes than internal fixation in terms of reoperation rate, complications, and postoperation pain. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Comparison of three treatment methods for simple bone cyst in children.

BACKGROUND: The unicameral bone cyst (UBC) is a kind of benign tumor whose clinical treatments and efficacy are controversial. The purpose of this study was to evaluate the efficacy of the elastic stable intramedullary nail (ESIN), the injection of autologous bone marrow (ABM), and the combination of ESIN and ABM in the treatment of bone cyst in children. METHODS: Eighty-three cases with simple bone cyst were analyzed retrospectively. Twenty-eight cases were treated with ABM. Twenty-eight cases were treated with ESIN. Twenty-seven cases were treated with ABM and ESIN. All cases were diagnosed through X-ray, CT, or MRI scans. For the suspicious ones, the pathological biopsy was performed for an accurate diagnosis. X-ray examinations were carried out for the postoperative follow-up. Capanna criteria for bone cyst was used for postoperative evaluation of three methods. RESULTS: All cases accomplished the follow-up. The effective rate of the ABM + ESIN group was significantly higher than that of the ABM group (P < 0.05), and the cure rates of the ESIN group and the ABM + ESIN group were higher than that of the ABM group (P < 0.05, respectively). The cure time in the ESIN group was lower than that of the other two groups (P < 0.05, respectively). The times for admission were 2.0 ± 0.0 in the ESIN group, 5.7 ± 1.9 in the ABM group, and 4.7 ± 2.4 in the ABM + ESIN group (P < 0.05 when compared with each other). CONCLUSIONS: The method of ABM combined with ESIN for children's bone cyst has the highest effective rate and curative rate. For the individual method, ESIN has a higher effective rate and curative rate than that of ABM. Meanwhile, it has the fewest time of hospitalization.

Epidural electrical stimulation effectively restores locomotion function in rats with complete spinal cord injury.

Epidural electrical stimulation can restore limb motor function after spinal cord injury by reactivating the surviving neural circuits. In previous epidural electrical stimulation studies, single electrode sites and continuous tetanic stimulation have often been used. With this stimulation, the body is prone to declines in tolerance and locomotion coordination. In the present study, rat models of complete spinal cord injury were established by vertically cutting the spinal cord at the T8 level to eliminate disturbance from residual nerve fibers, and were then subjected to epidural electrical stimulation. The flexible extradural electrode had good anatomical topology and matched the shape of the spinal canal of the implanted segment. Simultaneously, the electrode stimulation site was able to be accurately applied to the L2-3 and S1 segments of the spinal cord. To evaluate the biocompatibility of the implanted epidural electrical stimulation electrodes, GFAP/Iba-1 double-labeled immunofluorescence staining was performed on the spinal cord below the electrodes at 7 days after the electrode implantation. Immunofluorescence results revealed no significant differences in the numbers or morphologies of microglia and astrocytes in the spinal cord after electrode implantation, and there was no activated Iba-1+ cell aggregation, indicating that the implant did not cause an inflammatory response in the spinal cord. Rat gait analysis showed that, at 3 days after surgery, gait became coordinated in rats with spinal cord injury under burst stimulation. The regained locomotion could clearly distinguish the support phase and the swing phase and dynamically adjust with the frequency of stimulus distribution. To evaluate the matching degree between the flexible epidural electrode (including three stimulation contacts), vertebral morphology, and the level of the epidural site of the stimulation electrode, micro-CT was used to scan the thoracolumbar vertebrae of rats before and after electrode implantation. Based on the experimental results of gait recovery using three-site stimulation electrodes at L2-3 and S1 combined with burst stimulation in a rat model of spinal cord injury, epidural electrical stimulation is a promising protocol that needs to be further explored. This study was approved by the Animal Ethics Committee of Chinese PLA General Hospital (approval No. 2019-X15-39) on April 19, 2019.

Astrocytes directly clear myelin debris through endocytosis pathways and followed by excessive gliosis after spinal cord injury.

Persisted myelin debris inhibit axon regeneration and contribute to further tissue damage after spinal cord injury (SCI). The traditional view is that myelin debris is mainly cleared by microglia and macrophages, while astrocytes cannot directly engulf myelin debris because they are absent from lesion core. Here, we definitely showed that astrocytes could directly uptake myelin debris both in vitro and in vivo to effectively complement the clearance function. Therefore, it can be shown that astrocytes can exert myelin clearance effect directly and indirectly after spinal cord injury. The damaged myelin debris was transported to lysosomes for degradation through endocytosis pathways, finally resulting in excessive gliosis. This process may be a potential target for regulating neural tissue repair and excessive glia scar formation after SCI.

Correction: Mutation spectrums of TSC1 and TSC2 in Chinese women with lymphangioleiomyomatosis (LAM).

[This corrects the article DOI: 10.1371/journal.pone.0226400.].

Georhizobium profundi gen. nov., sp. nov., a piezotolerant bacterium isolated from a deep-sea sediment sample of the New Britain Trench.

A novel alphaproteobacterium, strain WS11T, was isolated from a deep-sea sediment sample collected from the New Britain Trench. The full-length 16S rRNA gene of strain WS11T had the highest sequence similarity of 97.6 % to Rhizobium subbaraonis JC85T, followed by Mycoplana ramosa DSM 7292T (96.9 %) and Rhizobium azooxidifex Po 20/26T (96.8 %). Phylogenetic analysis of concatenated 16S rRNA, atpD and recA gene sequences showed that strain WS11T was deeply separated from the species within the family Rhizobiaceae. Phylogenomic analysis based on the whole-genome protein sequences showed that strain WS11T formed an independent monophyletic branch in the family Rhizobiaceae, paralleled with the species in the families Brucellaceae and Phyllobacteriaceae within the order Rhizobiales. Cells were Gram-stain-negative, oxidase- and catalase-positive, and aerobic short rods (1.5-2.4×0.9-1.0 µm). Growth was observed at salinities ranging from 0 to 5% (optimum, 1 %), from pH 6.5 to 9 (optimum, pH 7) and at temperatures between 20 and 30 °C (optimum, 28 °C). Strain WS11T was piezotolerant, growing optimally at 0.1 MPa (range 0.1-70 MPa). The main fatty acid was summed feature 8 (C18 : 1 ω7c/C18  : 1 ω 6c). The sole respiratory quinone was ubiquinone-10 (Q-10). The predominant polar lipids were phosphatidylcholine, two unidentified aminophospholipids and an unidentified phospholipid. The genome size was about 4.36 Mbp and the G+C content was 62.3 mol%. The combined genotypic and phenotypic data show that strain WS11T represents a novel species of a novel genus in the family Rhizobiaceae, for which the name Georhizobium profundi gen. nov., sp. nov. is proposed (type strain WS11T=MCCC 1K03498T=KCTC 62439T).

Mutation spectrums of TSC1 and TSC2 in Chinese women with lymphangioleiomyomatosis (LAM).

The aim of our study was to elucidate the landscapes of genetic alterations of TSC1 and TSC2 as well as other possible non-TSC1/2 in Lymphangioleiomyomatosis (LAM) patients. Sixty-one Chinese LAM patients' clinical information was collected. Tumor biopsies and matched leukocytes from these patients were retrospectively analyzed by next generation sequencing (NGS), chromosomal microarray analysis (CMA), and multiplex ligation-dependent probe amplification (MLPA). Eighty-six TSC1/2 variants were identified in 46 of the 61 LAM patients (75.4%) in which TSC2 and TSC1 variants were 88.37% and 11.63% respectively. The 86 variants are composed of (i) 52 single nucleotide variants (SNVs) (including 30 novel variants), (ii) 23 indels (including 21deletions, and 2 insertions), (iii) a germline duplication of exon 31-42 of TSC2, (iv) a 2.68 Mb somatic duplication containing TSC2, and (v) 9 regions with copy-neutral loss of heterogeneity (CN-LOHs) present only in the LAM patients with single TSC1/2 mutations. Sixty-one non-TSC1/2 variants in 31 genes were identified in 37 LAM patients. Combined applications of different techniques are necessary to achieve maximal detection rate of TSC1/2 variants in LAM patients. Thirty novel TSC1/2 variants expands the spectrum of TSC1/2 in LAM patients. Identification of 61 non-TSC1/2 variants suggests that alternative genes might have contributed to the initiation and progression of LAM.