Xinlong Ma

Comparison of Decellularization Protocols for Preparing a Decellularized Porcine Annulus Fibrosus Scaffold

Tissue-specific extracellular matrix plays an important role in promoting tissue regeneration and repair. We hypothesized that decellularized annular fibrosus matrix may be an appropriate scaffold for annular fibrosus tissue engineering. We aimed to determine the optimal decellularization method suitable for annular fibrosus. Annular fibrosus tissue was treated with 3 different protocols with Triton X-100, sodium dodecyl sulfate (SDS) and trypsin. After the decellularization process, we examined cell removal and preservation of the matrix components, microstructure and mechanical function with the treatments to determine which method is more efficient. All 3 protocols achieved decellularization; however, SDS or trypsin disturbed the structure of the annular fibrosus. All protocols maintained collagen content, but glycosaminoglycan content was lost to different degrees, with the highest content with TritonX-100 treatment. Furthermore, SDS decreased the tensile mechanical property of annular fibrosus as compared with the other 2 protocols. MTT assay revealed that the decellularized annular fibrosus was not cytotoxic. Annular fibrosus cells seeded into the scaffold showed good viability. The Triton X-100–treated annular fibrosus retained major extracellular matrix components after thorough cell removal and preserved the concentric lamellar structure and tensile mechanical properties. As well, it possessed favorable biocompatibility, so it may be a suitable candidate as a scaffold for annular fibrosus tissue engineering.

Integrated trilayered silk fibroin scaffold for osteochondral differentiation of adipose-derived stem cells.

Repairing osteochondral defects (OCD) remains a formidable challenge due to the high complexity of native osteochondral tissue and the limited self-repair capability of cartilage. Osteochondral tissue engineering is a promising strategy for the treatment of OCD. In this study, we fabricated a novel integrated trilayered scaffold using silk fibroin and hydroxyapatite by combining paraffin-sphere leaching with a modified temperature gradient-guided thermal-induced phase separation (TIPS) technique. This biomimetic scaffold is characterized by three layers: a chondral layer with a longitudinally oriented microtubular structure, a bony layer with a 3D porous structure and an intermediate layer with a dense structure. Live/dead and CCK-8 tests indicated that this scaffold possesses good biocompatibility for supporting the growth, proliferation, and infiltration of adipose-derived stem cells (ADSCs). Histological and immunohistochemical stainings and real-time polymerase chain reaction (RT-PCR) confirmed that the ADSCs could be induced to differentiate toward chondrocytes or osteoblasts in vitro at chondral and bony layers in the presence of chondrogenic- or osteogenic-induced culture medium, respectively. Moreover, the intermediate layer could play an isolating role for preventing the cells within the chondral and bony layers from mixing with each other. In conclusion, the trilayered and integrated osteochondral scaffolds can effectively support cartilage and bone tissue generation in vitro and are potentially applicable for OC tissue engineering in vivo.

Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor.

Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration.

Silk fibroin porous scaffolds for nucleus pulposus tissue engineering.

Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38±5.12% and pore size of 165.00±8.25μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell-scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue.

Intervertebral Disc Tissue Engineering with Natural Extracellular Matrix-Derived Biphasic Composite Scaffolds

Tissue engineering has provided an alternative therapeutic possibility for degenerative disc diseases. However, we lack an ideal scaffold for IVD tissue engineering. The goal of this study is to fabricate a novel biomimetic biphasic scaffold for IVD tissue engineering and evaluate the feasibility of developing tissue-engineered IVD in vitro and in vivo. In present study we developed a novel integrated biphasic IVD scaffold using a simple freeze-drying and cross-linking technique of pig bone matrix gelatin (BMG) for the outer annulus fibrosus (AF) phase and pig acellular cartilage ECM (ACECM) for the inner nucleus pulposus (NP) phase. Histology and SEM results indicated no residual cells remaining in the scaffold that featured an interconnected porous microstructure (pore size of AF and NP phase 401.4±13.1 μm and 231.6±57.2 μm, respectively). PKH26-labeled AF and NP cells were seeded into the scaffold and cultured in vitro. SEM confirmed that seeded cells could anchor onto the scaffold. Live/dead staining showed that live cells (green fluorescence) were distributed in the scaffold, with no dead cells (red fluorescence) being found. The cell—scaffold constructs were implanted subcutaneously into nude mice and cultured for 6 weeks in vivo. IVD-like tissue formed in nude mice as confirmed by histology. Cells in hybrid constructs originated from PKH26-labeled cells, as confirmed by in vivo fluorescence imaging system. In conclusion, the study demonstrates the feasibility of developing a tissue-engineered IVD in vivo with a BMG- and ACECM-derived integrated AF-NP biphasic scaffold. As well, PKH26 fluorescent labeling with in vivo fluorescent imaging can be used to track cells and analyse cell—scaffold constructs in vivo.

Involvement of periostin-sclerostin-Wnt/β-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin.

Periostin has an essential role in mechanotransduction in bone. Naringin, a natural flavonoid, has been evidenced for its osteoprotective role in osteoporosis, while its mechanism is far from clear. Here we show that down-regulation of periostin, and up-regulation of its downstream sclerostin and inactivation of Wnt/β-catenin signaling were implicated in neurectomy-induced bone loss. Naringin could up-regulate periostin and prevent neurectomy-induced deterioration of BMD, trabecular microstructure and bone mechanical characteristics. In conclusion, naringin could prevent progress of disuse osteoporosis in rats, which may be mediated by increased periostin expression and subsequently inhibition of sclerostin and activation of Wnt/β-catenin signaling pathways.

Efficiency and Safety of Intravenous Tranexamic Acid in Simultaneous Bilateral Total Knee Arthroplasty: A Systematic Review and Meta-analysis

The objective of this systematic review and meta‐analysis was to evaluate the efficacy and safety of i.v. tranexamic acid (TXA) in simultaneous bilateral total knee arthroplasty (TKA). Potentially relevant published reports were identified from the following electronic databases: Medline, PubMed, Embase, ScienceDirect and Cochrane Library. RevMan v5.3was used to pool data. Two randomized controlled trials and four case‐control studies met the inclusion criteria. The current meta‐analysis identified significant differences between TXA group and control groups in terms of postoperative hemoglobin concentration (P < 0.01), drainage volume (P < 0.01), transfusion rate (P < 0.01) and units transfused (P = 0.006). There were no significant differences in length of stay (P = 0.66), operation time (P = 0.81) or and incidence of adverse effects such as infection (P = 0.42), deep venous thrombosis (DVT) (P = 0.88) and pulmonary embolism (PE) (P = 0.11). Our results show that i.v. administration of TXA in simultaneous bilateral TKA reduces postoperative drops in hemoglobin concentration, drainage volume, and transfusion requirements and does not prolong length of stay or operation time. Moreover, no adverse effects, such as infection, DVT or PE, were associated with TXA.

Novel Cartilage-derived Biomimetic Scaffold for Human Nucleus Pulposus Regeneration: a Promising Therapeutic Strategy for Symptomatic Degenerative Disc Diseases

Because current therapies have not always been successful and effective, the possibility of regenerating the nucleus pulposus (NP) through a tissue‐engineered construct offers a novel therapeutic possibility for symptomatic degenerative disc diseases (DDDs). However, more research is necessary to identify the optimal scaffold, cell type and mixture of signal factors needed for NP regeneration. Numerous possible scaffolds for NP regeneration have been investigated; they have many shortcomings in common. Various biological scaffolds derived from decellularized tissue and organs have been successfully used in tissue engineering and received approval for use in humans. Regretfully, harvesting of human NP is difficult and only small amounts can be obtained. The macromolecules of cartilage, which include collagen and proteoglycan aggrecan, are similar to those of the extracellular matrix of immature NP. Recent studies have shown that adipose‐derived stem cells (ADSC) can be induced to develop NP‐like phenotypes when stimulated by appropriate signals. We thus reasonably postulated that an ideal NP scaffold for tissue engineering could be fabricated from decellularized cartilage matrix (DCM). Furthermore, a combination of ADSCs and DCM‐derived biomimetic scaffolds would be advantageous in NP tissue engineering and, in the long run, could become an effective treatment option for symptomatic DDD.

Circumferentially oriented microfiber scaffold prepared by wet-spinning for tissue engineering of annulus fibrosus

Repairing damaged annulus fibrosus (AF) is one of the most challenging topics for treating intervertebral disc (IVD) disease. Tissue engineering combining scaffolds with cells provides a promising solution. However, fabricating scaffolds with a circumferentially oriented fibrous structure similar to native AF remains a big challenge. In this study, we present an effective and convenient wet spinning strategy for fabricating an AF scaffold composed of circumferentially oriented poly(e-caprolactone) microfibers. Cell culture experiments demonstrated that this scaffold could support AF cell attachment, proliferation and infiltration as confirmed by scanning electron microscopy (SEM), confocal microscopy, live/dead staining and a MTT assay, respectively. Histological, immunohistochemical staining, biochemical quantitative analysis and RT-PCR showed that the AF cells (AFCs) inside scaffolds could spread along the microfiber direction and secrete an AF-related extracellular matrix (e.g., glycosaminoglycans, collagen type I and II) which also oriented along the microfiber direction. As a result, the compressive and tensile properties were enhanced with increasing culture time. These results demonstrate the feasibility of using this new wet-spun microfibrous oriented scaffold for AFCs culture, and the potential application for regeneration of AF.

Radiographic measurement for transforaminal percutaneous endoscopic approach (PELD)

ObjectiveA radiographic study to analyze the working zone and relationship of the nerve root to their corresponding intervertebral disc for transforaminal percutaneous approaches.Methods100 MRIs of transverse and sagittal views of 37 males, 63 females (average age 45xa0years), 50 MRIs of coronal views of 22 males, 28 females (average age 42xa0years), and 100 X-rays, 46 males, 54 females (average age of 44xa0years) were used for image analysis. All radiologic measurements were obtained independently by two experienced radiologists. On sagittal plane, foraminal height, foraminal diameter, nerve root-disc distance and nerve root-pedicle distance were measured. On transverse plane, foraminal width, nerve root-disc distance, nerve root-facet distance and target angle (J°) were analyzed at the superior (s) and inferior (i) margin of the disc. On coronal plane, nerve root-disc distance and nerve root-pedicle distance were measured at the medial, middle and lateral borders of the pedicle.ResultsSagittal plane; foraminal height and diameter decreased caudally. Transverse plane; foraminal width was larger at the superior margin of the disc. Nerve root-disc distance decreased caudally. The nerve root lied dorsal to the disc at L2–L3 and L3–L4, whereas at L4–L5 and L5–S1 it lied ventrally. Nerve root-facet distance was shortest at the superior margin. Target angles (Js°, Ji°) at L2–L3 and L3–L4 were wider at their superior margin than at their inferior margin. Coronal plane; nerve root-disc distance increased from L2–L3 to L5–S1 whereas nerve root-pedicle distances decreased, thus coursing more vertically.ConclusionsAt lower lumbar levels the exiting nerve root is at risks of injury. Hence, it is advised to enlarge the foramen for safe passage of endoscopic instruments and to minimize the possibility of nerve injury.