Preparation of PLCL/ECM nerve conduits by electrostatic spinning technique and evaluationin vitroandin vivo.

Objective. Artificial nerve scaffolds composed of polymers have attracted great attention as an alternative for autologous nerve grafts recently. Due to their poor bioactivity, satisfactory nerve repair could not be achieved. To solve this problem, we introduced extracellular matrix (ECM) to optimize the materials.Approach.In this study, the ECM extracted from porcine nerves was mixed with Poly(L-Lactide-co-ϵ-caprolactone) (PLCL), and the innovative PLCL/ECM nerve repair conduits were prepared by electrostatic spinning technology. The novel conduits were characterized by scanning electron microscopy (SEM), tensile properties, and suture retention strength test for micromorphology and mechanical strength. The biosafety and biocompatibility of PLCL/ECM nerve conduits were evaluated by cytotoxicity assay with Mouse fibroblast cells and cell adhesion assay with RSC 96 cells, and the effects of PLCL/ECM nerve conduits on the gene expression in Schwann cells was analyzed by real-time polymerase chain reaction (RT-PCR). Moreover, a 10 mm rat (Male Wistar rat) sciatic defect was bridged with a PLCL/ECM nerve conduit, and nerve regeneration was evaluated by walking track, mid-shank circumference, electrophysiology, and histomorphology analyses.Main results.The results showed that PLCL/ECM conduits have similar microstructure and mechanical strength compared with PLCL conduits. The cytotoxicity assay demonstrates better biosafety and biocompatibility of PLCL/ECM nerve conduits. And the cell adhesion assay further verifies that the addition of ECM is more beneficial to cell adhesion and proliferation. RT-PCR showed that the PLCL/ECM nerve conduit was more favorable to the gene expression of functional proteins of Schwann cells. Thein vivoresults indicated that PLCL/ECM nerve conduits possess excellent biocompatibility and exhibit a superior capacity to promote peripheral nerve repair.Significance.The addition of ECM significantly improved the biocompatibility and bioactivity of PLCL, while the PLCL/ECM nerve conduit gained the appropriate mechanical strength from PLCL, which has great potential for clinical repair of peripheral nerve injuries.

[Application Effect of Continuous Lumbar Cistern Fluid Drainage Combined With Decompressive Craniectomy in the Treatment of Severe Craniocerebral Injury].

Objective: To analyze the application effect of continuous lumbar cistern fluid drainage combined with decompressive craniectomy in the treatment of severe craniocerebral injury. Methods: A total of 87 patients with severe craniocerebral injury admitted to our hospital between March 2016 and March 2021 were retrospectively enrolled. They were divided into two groups according to the decompression methods applied, with 42 patients who received standard decompressive craniectomy assigned to the control group and 45 patients who received continuous lumbar cistern fluid drainage combined with standard decompressive craniectomy assigned to the observation group. The primary indicators that were monitored and compared between the two group included the amount of time for patient CT imaging to be clear of subarachnoid hemorrhage, the length-of-stay, the duration of post-operative intubation, the mannitol dose, scores for Glasgow Coma Scale (GCS), prognosis, the incidence of cerebral edema and cerebral infarction, and complications. The secondary indicators that were monitored and compared included intracranial pressure, cerebrospinal fluid antinucleosome protein SP100, and red blood cell count of the two groups before treatment and after continuous drainage for 7 days. Results: The amount of time for CT imaging to be clear of subarachnoid hemorrhage and the length-of-stay of the observation group were shorter than those of the control group, the mannitol dose of the observation group was lower than that of the control group, the incidence of cerebral edema and the incidence of complications of the observation group were lower than those of the control group, and the rate of patients with good prognosis in the observation group was higher than that in the control group ( P<0.05). There was no significant difference in the rate of poor prognosis or mortality between the two groups ( P>0.05). The duration of postoperative intubation of the observation group was (8.24±1.09) d, while that of the control group was (9.22±1.26) d, and the difference between the two groups was statistically significant ( t=3.887, P<0.05). There were 2 cases (4.44%) of cerebral infarction in the observation group, with the infarct volume being (8.36±1.87) cm 3, while there were 9 cases (21.43%) of cerebral infarction in the control group, with the infarct volume being (8.36±1.87) cm 3, and there were statistically significant differences in the incidence and volume of cerebral infarction between the two groups ( χ 2=5.674, t=9.609, P<0.05). After treatment, the intracranial pressure and red blood cell count decreased in both groups and the intracranial pressure, cerebrospinal fluid SP100, and red blood cell count of the observation group were significantly lower than those of the control group ( P<0.05). The cerebrospinal fluid SP100 of the observation group decreased after treatment in comparison with the level before treatment ( P<0.05), while the pre- and post-treatment levels of the control group did not demonstrate any significant difference. Conclusion: Continuous lumbar cistern fluid drainage in patients with severe craniocerebral injury effectively shortens the time required for the body to recover, significantly reduces the level of intracranial pressure, improves the levels of cerebral edema and cerebral infarction, and has a high degree of safety for prognosis and recovery.

Decellularized tendon matrix membranes prevent post-surgical tendon adhesion and promote functional repair.

Adhesion often occurs after tendon injury, and results in sliding disorder and movement limitation with no ideal solution for it in clinic. In this study, an anti-adhesion membrane, i.e., decellularized tendon matrix (DTM) for tendon is successfully prepared by an optimized tendon decellularization method from homologous extracellular matrix. Microsection technology has been used to optimize the method of decellularization in order to better preserve the bioactive components in tissues and reduce the chemical reagent residues on the premise of effective decellularization with relatively shorter time and less reagents for decellularization. The physic-chemical properties and biological functions of DTM are evaluated, and high-throughput and high-precision tandem mass tags (TMT) labeling proteomics technology is used to analyze protein components of DTM, which may provide the scientific support for application of the innovative product. In vitro biosafety tests show that DTM not only is non-toxic but also promote cell proliferation. Subcutaneous implantation test confirms that DTM is completely degraded after 12 weeks and there is no obvious inflammatory reaction. The results of Achilles tendon repair in rabbits show that DTM can not only prevent tendon adhesion but also improve the quality of tendon repair, which demonstrates its tremendous application potential. STATEMENT OF SIGNIFICANCE: There is no ideal solution for adhesion after tendon injury. In this study, a dense tendon anti-adhesion membrane (DTM) was successfully prepared from homologous extracellular matrix (ECM). This DTM could effectively retain bioactive ingredients, and prevent adhesion as well as improve the quality of tendon repair in vivo. An optimized decellularization method was used which could effectively decellularize tendon in a short time, better preserve bioactive components, and reduce reagent residues. For the first time, high-throughput and high-precision tandem mass tags (TMT) labeling proteomics technology was used to qualitatively and quantitatively analyze the protein composition of fresh tendon, acellular tendon and DTM, which provided not only scientific support for the application of DTM, but also comprehensive and accurate data support for related research of bovine tendons and decellularization.

Fabrication and evaluation of an optimized acellular nerve allograft with multiple axial channels.

Acellular nerve allografts are promising alternatives to autologous nerve grafts, but still have many drawbacks which greatly limit their curative effects. Here, we developed an optimized acellular nerve allograft with multiple axial channels by a modified decellularization method. These allografts were confirmed to preserve more extracellular matrix components and factors, and remove cellular components effectively. Meanwhile, macrochannels and microchannels were introduced to optimize internal microstructure of allografts, which increases porosity and water absorption, without significant loss of mechanical strength. The in vitro experiments demonstrated that the multichannel allografts showed superior ability of facilitating proliferation and penetration of Schwann cells. Additionally, in the in vivo experiments, the multichannel allografts were used to bridge 10 mm rat sciatic nerve defects. They exhibited better capacity to guide regenerative nerve fibers through the defective segment and restore innervation of target organs, thus achieving better recovery of muscle and motor function, in comparison with conventional acellular allografts. These findings indicate that this multichannel acellular nerve allograft has great potential for clinical application and provides a new prospective for future investigations of nerve regeneration. STATEMENT OF SIGNIFICANCE: Acellular nerve allografts, with preservation of natural extracellular matrix, are officially approved to repair peripheral nerve injury in some countries. However, bioactive component loss and compact internal structure result in variable clinical effects of conventional acellular allografts. In the present study, we fabricated an optimized acellular nerve allograft with multiple axial channels, which could both enable decellularization to be easily accomplished and reduce the amount of detergents in the preparation process. Characterization of the multichannel acellular allografts was confirmed to have better preservation of ECM bioactive molecules and regenerative factors. Efficiency evaluation showed the multichannel allografts could facilitate Schwann cells to migrate inside them in vitro, and enhance regrowth and myelination of axons as well as recovery of muscle and motor function in vivo.

Preparation and evaluation of acellular sheep periostea for guided bone regeneration.

The objective of this study was to fabricate an acellular sheep periosteum and explore its potential application in guided bone regeneration. Sheep periosteum was collected and decellularized by a modified decellularization protocol. The effectiveness of cell removal was proved by hematoxylin and eosin and 4',6-diamidino-2-phenylindole staining, DNA quantitative test, and agarose gel electrophoresis. After decellularization, its microstructure was found to become more porous while the integrality of collagen fibers remained undamaged, and the contents of collagen and glycosaminoglycan were not decreased significantly. Biomechanical analysis showed that the elastic modulus was significantly declined, while the yield stress was not affected, probably due to the collagen integrality. In vitro study of CCK-8 assay demonstrated that the acellular periosteum not only had no toxic effect to the MC3T3-E1 cells, but benefited the cell proliferation to some degree. In vivo experiment of guided bone regeneration was performed using a rabbit cranial model. Micro-CT and histological results revealed that the acellular periosteum not only effectively prevented the ingrowth of fibrous connective tissues, but also potentially facilitated bone regeneration. In conclusion, acellular sheep periostea, with wider sources, less costs, and more convenient fabrication process, would have great potential in the employment for guided bone regeneration.

In vitro and in vivo biocompatibility study on acellular sheep periosteum for guided bone regeneration.

This study addresses the fabrication of an extracellular matrix material of the acellular sheep periosteum and the systematic evaluation of its biocompatibility to explore its potential application in guided bone regeneration. Sheep periosteum was harvested and decellularized by a combined decellularization protocol. The effectiveness of cell removal was proved and residual α-Gal antigen was also quantitatively detected. Then, mouse MC3T3-E1 cells were seeded onto the acellular periosteum. A scanning electron microscope (SEM) was used to record the whole process of cell adhesion. The CCK-8 assay suggested that the acellular periosteum not only had zero toxic effect on pre-osteoblasts, but played a positive role in cell proliferation. We also tested whether the acellular periosteum possesses favorable osteogenesis induction activity using an alkaline phosphatase (ALP) assay and a quantitative real-time PCR (Col I, Runx2, OCN) assay. An in vivo study of a subcutaneous implantation test using Sprague Dawley (SD) rats was performed to detect the changes in IL-2, IFN-γ and IL-4 in serum and elucidate the host's local response to acellular periosteum through hematoxylin and eosin (HE) and immunohistochemical staining. The results show that acellular sheep periosteum did not elicit a severe immunogenic response via the Th1 pathway, unlike fresh sheep periosteum. In conclusion, acellular sheep periosteum possesses favorable biocompatibility to be employed for guided bone regeneration.