Objective To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility. Methods The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats. Results The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded. Conclusion The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.
Objective Native extracellular matrix (ECM) is comprised of a complex network of structural and regulatory proteins that are arrayed into a tissue-specific, biomechanically optimal, fibrous matrix. The multifunctional nature of the native ECM will need to be considered in the design and fabrication of tissue engineering scaffolds. To investigate the extraction techniques of naturally derived nerve ECM and the feasibil ity of nerve tissue engineering scaffold. Methods Ten fresh canine sciatic nerves were harvested; nerve ECM material was prepared by hypotonic freeze-thawing, mechanicalgrinding, and differential centrifugation. The ECM was observed by scanning electron microscope. Immunofluorescencestaining was performed to detect specific ECM proteins including collagen type I, laminin, and fibronectin. Total collagen and glycosaminoglycan (GAG) contents were assessed using biochemical assays. The degree of decellularization was evaluated with staining for nuclei using Hoechst33258. The dorsal root gangl ion and Schwann cells of rats were respectively seeded onto nerve tissue-specific ECM films. The biocompatibil ity was observed by specific antibodies for cell markers. Results Scanning electron microscope analysis revealed that nerve-derived ECM consisted of a nanofibrous structure, which diameter was 30-130 nm. Immunofluorescence staining confirmed that the nerve-derived ECM was made up of collagen type I, laminin, and fibronectin. The histological staining showed that the staining results of sirius red, Safranin O, and toluidine blue were positive. Hoechst33258 staining showed no DNA within the decellularized ECM. Those ECM films had good biocompatibil ity for dorsal root gangl ion and Schwann cells. The cotents of total collagen and GAG in the nerve-derived ECM were (114.88 ± 13.33) μg/ mg and (17.52 ± 2.34) μg/mg, showing significant difference in the content of total collagen (P lt; 0.01) and no significant difference in the content of GAG (P gt; 0.05) when compared with the contents of normal nerve tissue [(54.07 ± 5.06) μg/mg and (25.25 ± 1.56) μg/mg)]. The results of immunofluorescence staining were positive for neurofilament 200 after 7 days and for S100 after 2 days. Conclusion Nerve-derived ECM is rich in collagen type I, laminin, and fibronectin and has good biocompatibil ity, so it can be used as a nerve tissue engineering scaffold.
Objective To explore the effect of controlled release of nerve growth factor (NGF) on peripheral nerve defect repaire by acellular nerve graft. Methods The microspheres of NGF were prepared with drug microsphere technologyand fixed with the fibrin glue to make the compl icated controlled release NGF. Twenty healthy male SD rats weighing 280-300 g were adopted to prepare acellular xenogenous nerve, 52 male Wistar rats weighing 250-300 g were adopted to prepare the 10 mm defect model of left sciatic nerve. and thereafter were randomly divided into 4 groups: autograft group(group A), acellular nerve allograft combined with the double controlled release NGF (group B), acellular nerve allograft (group C) and acellular nerve allograft combined with fibrin glue (group D). Without any operation, the right sciatic nerve was regarded as control group. General observation was conducted after operation. The nerve axon regeneration length was measured 2 weeks after operation. The effects of peripheral nerve regeneration were evaluated by neural electrophysiology, the recovery rate of triceps surae muscular tension and weight and histological assessment 16 weeks after operation. Results All the animals survived till the end of experiment. The length of nerve regeneration was measured at 2 weeks after transplantation. The regeneration nerve of group A was longer than that of other groups (P lt; 0.05), group B longer than groups C and D (P lt; 0.05), and there were no difference between group C and group D (P gt; 0.05). At 16 weeks after operation, the recovery rates of nerve conduction velocity of groups A and B (73.37% ± 7.82% and 70.39% ± 8.45%) were larger than that of groups C and D (53.51% ± 6.31% and 55.28% ± 5.37%) (P lt; 0.05). The recovery rates of the triceps surae muscular tension in group A (85.33% ± 5.59%) were larger than that in groups B, C and D (69.79% ± 5.31%, 64.46% ± 8.49％ and 63.35% ± 6.40%) (P lt; 0.05). There were no significant differences among groups B, C and D (P gt; 0.05). The recovery rates of the triceps surae weight in group A (62.54% ± 8.25%) werelarger than that in groups B, C and D (53.73% ± 4.56%, 46.37% ± 5.68% and 45.78% ± 7.14%, P lt; 0.05). There was significant difference between group B and groups C, D (P lt; 0.05) and no significant differences between group C and group D (P gt; 0.05). The histological observation indicated that axon number and myel in thickness in group B were larger than those in group C and group D (P lt; 0.05). The axonal diameter in group B was significantly less than that in group A (P lt; 0.05). Conclusion Acellular nerve graft combined with the controlled release NGF is a satisfactory alternative to repair the peripheral nerve defect.
Objective Using chemically extracted acellular methods to treat extracranial section of the canine whole facial nerve, to evaluated its effects on nerve structure and the removal extent of Schwann cells and myel in. Methods Twenty whole facial nerves were exposed from 10 canines [weighing (18 ± 3) kg]. The extracranial trunk of canine facial nerve and its branches (temporal branch, zygomatic branch, buccal branch, marginal mandibular branch, and cervical branch) were dissected under l ight microscope. Twenty facial nerves were divided into the experimental group (n=12) and control group (n=8) randomly. In experimental group, the nerve was extracted with the 3%TritonX-100 and 4% sodium deoxycholate. In control group, the nerve was not extracted. HE staining and immunofluorescence histological stainings for Hoechst33258, P75, Zero, and Laminin were performed. Results After histological staining, it was found that myel in and Schwann cells were removed from the facial nerve while the basal lamina tube remained intact. The whole canine facial nerves (one nerve trunk and multiple nerve branches) had the similar result. Conclusion The canine whole facial nerve has natural structure (one nerve trunk and multiple nerve branches) by extracted with chemically extracted acellular methods, so it is an available graft for repairing the defect of the whole facial nerve.
ObjectiveElectrospinning technique was used to manufacture polycaprolactone (PCL)/collagen typeⅠ nanofibers orientated patches and to study their physical and chemical characterization, discussing their feasibility as synthetic patches for rotator cuff repairing.MethodsPCL patches were prepared by electrospinning with 10% PCL electrospinning solution (control group) and PCL/collagen typeⅠorientated nanofibers patches were prepared by electrospinning with PCL electrospinning solution with 25% collagen type Ⅰ(experimental group). The morphology and microstructure of the two patches were observed by gross and scanning electron microscopy, and the diameter and porosity of the fibers were measured; the mechanical properties of the patches were tested by uniaxial tensile test; the composition of the patches was analyzed by Fourier transform infrared spectroscopy; and the contact angle of the patch surface was measured. Two kinds of patch extracts were co-cultured with the third generation of rabbit tendon stem cells. Cell counting kit 8 (CCK-8) was used to detect the toxicity and cell proliferation of the materials. Normal cultured cells were used as blank control group. Rabbit tendon stem cells were co-cultured with the two patches and stained with dead/living cells after 3 days of in vitro culture, and laser confocal scanning microscopy was used to observe the cell adhesion and activity on the patch.ResultsGross and scanning electron microscopy showed that the two patch fibers were arranged in orientation. The diameter of patch fibers in the experimental group was significantly smaller than that in the control group (t=26.907, P=0.000), while the porosity in the experimental group was significantly larger than that in the control group (t=2.506, P=0.032). The tensile strength and Young’s modulus of the patch in the experimental group were significantly higher than those in the control group (t=3.705, P=0.029; t=4.064, P=0.034). Infrared spectrum analysis showed that PCL and collagen type Ⅰ were successfully mixed in the experimental group. The surface contact angle of the patch in the experimental group was (73.88±4.97)°, which was hydrophilic, while that in the control group was (128.46±5.10) °, which was hydrophobic. There was a significant difference in the surface contact angle between the two groups (t=21.705, P=0.002). CCK-8 test showed that with the prolongation of culture time, the cell absorbance (A) value increased gradually in each group, and there was no significant difference between the experimental group and the control group at each time point (P>0.05). Laser confocal scanning microscopy showed that rabbit tendon stem cells could adhere and grow on the surface of both patches after 3 days of culture. The number of cells adhered to the surface of the patches in the experimental group was more than that in the control group, and the activity was better.ConclusionPCL/ collagen type Ⅰ nanofibers orientated patch prepared by electrospinning technology has excellent physical and chemical properties, cell adhesion, and no cytotoxicity. It can be used as an ideal scaffold material in tendon tissue engineering for rotator cuff repair in the future.