OBJECTIVE: To prove the biological characteristics of spinal intervertebral fusion with allograft cortical ring plus autogenous cancellus from iliac bone (called as composite ring). METHODS: Eight hybrid dogs were adopted in the study. The composite ring and autogenous tri-cortical blocks were implanted into the intervertebral space of dogs respectively. The intervertebral discs were removed before implantation. The fusion segments were evaluated by x-ray, histological examinations at 1, 2, 4 and 6 months after operation to compare the healing status of two implants. RESULTS: The X-ray film and histological examination showed the lumbar interbody was fused after 4 months of operation in composite ring group, while the autogenous iliac bone blocks fused with peripheral bone tissue after 6 months of operation. Composite ring healed more quickly and completely, showed different osteogenesis behavior compared with that of massive allografts. CONCLUSION: Composite ring can promote the fusion of intervertebral body and can be used as a potential material for spinal surgery.
Objective To investigate the promotion effect of neurotropic reinnervation with chemically extracted acellular nerve allograft. Methods The sciatic nerves of 5 healthy adult SD rats, regardless of gender and weighing 270-300 g, were collected to prepare chemically extracted acellular nerve allograft. Eighteen healthy adult Wistar rats, regardless of genderand weighing 300-320 g, were made the model of left sciatic nerve defect (10 mm) and randomly divided into 2 groups: autograft (control group, n=9) and allograft group (experimental group, n=9). The defects were bridged by acellular nerve allograft in experimental group and by autograft by turning over the proximal and distal ends of the nerve in control group. At 3 months after transplantation, dorsal root ganglion (DRG) resection operation was performed in 6 rats of 2 groups. At 3 weeks after operation, the sural nerves were harvested to calculate the misdirection rate of nerve fibers with pathological staining and compute-assisted image analysis. Cholinesterase staining and carbonic anhydrase staining were performed in the sural nerve of 3 rats that did not undergo DRG resection at 3 months. Results The results of chol inesterase staining and carbonic anhydrase staining showed that experimental group had less brown granules and more black granules than control group. After DRG resection, count of myelinated nerve fiber were 4 257 ± 285 in the experimental group and 4 494 ± 310 in the control group significant (P lt; 0.05). The misdirection rate was 2.27% ± 0.28% and 7.65% ± 0.68% in the experimental group and in the control group respectively, showing significant difference (P lt; 0.05). Conclusion Chemically extracted acellular nerve allograft can not only act as a scaffold in the period of nerve defects repair, but markedly enhance the effects of neurotropic reinnervation.
Objective Bone marrow mesenchymal stem cells (BMSCs), as replacement cells of Schwann cells, can increase the effect of peripheral nerve repair. However, it has not yet reached any agreement to add the appropriate number of seeded cells in nerve scaffold. To investigate the effect of different number of BMSCs on the growth of rat dorsal root gangl ia(DRG). Methods Three 4-week-old Sprague Dawley (SD) rats (weighing 80-100 g) were selected to isolate BMSCs, whichwere cultured in vitro. Three 1- to 2-day-old SD rats (weighing 4-6 g) were selected to prepare DRG. BMSCs at passage 3 were used to prepare BMSCs-fibrin glue complex. According to different number of BMSCs at passage 3 in fibrin glue, experiment was divided into group A (1 × 103), group B (1 × 104), group C (1 × 105), and group D (0, blank control), and BMSCs were cocultured with rat DRG. The axon length of DRG, Schwann cell migration distance, and axon area index were quantitatively evaluated by morphology, neurofilament 200, and Schwann cells S-100 immunofluorescence staining after cultured for 48 hours. Results Some long cell processes formed in BMSCs at 48 hours; migration of Schwann cells and axons growth from the DRG were observed, growing in every direction. BMSCs in fibrin glue had the biological activity and could effect DRG growth. The axon length of DRG and Schwann cell migration distance in groups A, B, and C were significantly greater than those in group D (P lt; 0.05). The axon length of DRG and Schwann cell migration distance in group C were significantly less than those in group B (P lt; 0.05), but there was no significant difference between group A and group C, and between group A and group B (P gt; 0.05). The axon area index in groups A and B was significantly greater than that in group D (P lt; 0.05), but there was no significant difference between group C and group D (P gt; 0.05); there was no significant difference in groups A, B, and C (P gt; 0.05). Conclusion In vitro study on DRG culture experiments is an ideal objective neural model of nerve regeneration. The effect of different number of BMSCs in fibrin glue on the growth of DRG has dose-effect relationship. It can provide a theoretical basis for the appropriate choice of the BMSCs number for tissue engineered nerve.
Objective Poly (propylene carbonate) (PPC), a newly reported polymer, has good biodegradabil ity and biocompatibil ity. To explore the feasibil ity of using electrospinning PPC materials in nerve tissue engineering, and to observe the effect of al igned and random PPC materials on axonal growth of rat dorsal root gangl ions (DRGs) in vitro. Methods Either al igned or randomly oriented sub-micron scale polymeric fiber was prepared with an electrospinning process. DRGs were harvested from 3 newborn Sprague-Dawley rats (female or male, weighing 4-6 g), and were incubated into 12-pore plate containing either al igned (the experimental group, n=6) or randomly oriented sub-micron scale polymeric fiber (the control group, n=6). The DRGs growth was observed with an inverted microscope; at 7 days immunofluorescent staining and scanning electronic microscope (SEM) observation were performed to quantify the extent of neurite growth andSchwann cells (SCs) migration. Results Either al igned or random fibers were fabricated by an electrospinning process. The diameter of the individual fiber ranged between 800 nm and 1 200 nm. In al igned PPC material, 90% fibers arranged in long axis direction, but the fibers in random PPC material arranged in all directions. The DRGs grew well in 2 PPC materials. Onthe al igned fiber film, the majority of neurite growth and SCs migration from the DRGs extended unidirectionally, parallel to the al igned fibers; however, neurite growth and SCs migration on the random fiber films oriented randomly. The extents of neurite growth were (2 684.7 ± 994.8) μm on the al igned fiber film and (504.7 ± 52.8) μm on the random fiber films, showing significant difference (t= —5.360, P=0.000). The distances of SCs migration were (2 770.6 ± 978.4) μm on the al igned fiber film and (610.2 ± 56.3) μm on the random fiber films, showing significant difference (t= —5.400, P=0.000). The extent of neurite growth was fewer than the distances of SCs migration in 2 groups. Conclusion The orientation structure of sub-micron scalefibers determines the orientation and extent of DRGs neurite growth and SCs migration. Al igned electrospinning PPC fiber is proved to be a promising biomaterial for nerve regeneration.
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.
ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.
Objective To observe the histological structure and cytocompatibility of novel acellular bone matrix (ACBM) and to investigate the feasibility as a scaffold for bone tissue engineering. Methods Cancellous bone columns were harvested from the density region of 18-24 months old male canine femoral head, then were dealt with high-pressure water washing, degreasing, and decellularization with Trixon X-100 and sodium deoxycholate to prepare the ACBM scaffold. The scaffolds were observed by scanning electron microscope (SEM); HE staining, Hoechst 33258 staining, and sirius red staining were used for histological analysis. Bone marrow mesenchymal stem cells (BMSCs) from canine were isolated and cultured with density gradient centrifugation; the 3rd passage BMSCs were seeded onto the scaffold. MTT test was done to assess the cytotoxicity of the scaffolds. The proliferation and differentiation of the cells on the scaffold were observed by inverted microscope, SEM, and live/dead cell staining method. Results HE staining and Hoechst 33258 staining showed that there was no cell fragments in the scaffolds; sirius red staining showed that the ACBM scaffold was stained crimson or red and yellow alternating. SEM observation revealed a three dimensional interconnected porous structure, which was the microstructure of normal cancellous bone. Cytotoxicity testing with MTT revealed no significant difference in absorbance (A) values between different extracts (25%, 50%, and 100%) and H-DMEM culture media (P gt; 0.05), indicating no cytotoxic effect of the scaffold on BMSCs. Inverted microscope, SEM, and histological analysis showed that three dimensional interconnected porous structure of the scaffold supported the proliferation and attachment of BMSCs, which secreted abundant extracellular matrices. Live/dead cell staining results of cell-scaffold composites revealed that the cells displaying green fluorescence were observed. Conclusion Novel ACBM scaffold can be used as an alternative cell-carrier for bone tissue engineering because of thoroughly decellularization, good mircostructure, non-toxicity, and good cytocompatibility.
Objective Surface modification of nitinol (NiTi) shape memory alloy is an available method to prevent nickel ion release and coating with titanium-niobium (TiNb) alloy will not affect the superelasticity and shape memory of NiTi. To evaluate the bone histocompatibil ity of NiTi shape memory alloy implants coated by TiNb in vivo. Methods NiTi memory alloy columns which were 4 mm in diameter and 12 mm in length were coated with Ti (Ti-coating group) and TiNb alloy (TiNb-coating group) respectively by magnetron sputtering technique. And NiTi group were not coated on the surface. Fifteen mongrel dogs were divided into 3 groups randomly with 5 dogs in each group. NiTi, Ti-coating and TiNb-coating columns were implanted into the lateral femoral cortex of each group, respectively. There were 10 columns embedded in eachdog’s femur whose distance was 1.0 cm to 1.5 cm from each other. The materials were obtained 12 months after operation. After X-ray photography, only those columns which were perpendicular to the cortex of the femur shaft were selected for subsequent analysis. Push-out tests were performed to attain the maximum shear strength (the number of specimens of TiNi group, Ticoating group, and TiNb-coating group were 12, 10, and 14, respectively). Undecalcified sections were used for histological observation and the calculation of osseointegration rate (the number of specimens of TiNi group, Ti-coating group, and TiNb-coating group were 8, 5, and 10, respectively). Results The maximum shear strength of Ti-coating group (95.10 ± 10.03) MPa, and TiNb-coating group (91.20 ± 15.42) MPa were significantly higher than that of NiTi group (71.60 ± 14.24) MPa (P lt; 0.01). Gimesa staining showed that no obvious macrophage and inflammation cell was observed in 3 groups. The osseointegration rates of NiTi group, Ti-coating group, and TiNb-coating group were (21.30% ± 0.23%), (32.50% ± 0.31%), and (38.60% ± 0.58%), respectively; there were significant differences among 3 groups (P lt; 0.01). Conclusion The implants of 3 groups all have good bone histocompatabil ity. But the osseointegration rate and the shear strength in the Ti-coating group and the TiNb-coating group were better than those in the NiTi group, the TiNb-coating group is the best among them.
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.
Objective To construct chemically extracted acellular nerve allograft (CEANA) with Schwann cells (SCs) from different tissues and to compare the effect of repairing peripheral nerve defect. Methods Bone marrow mesenchymal stem cells (BMSCs) and adi pose-derived stem cells (ADSCs) were isolated and cultured from 3 4-week-old SD mice with weighing 80-120 g. BMSCs and ADSCs were induced to differentiated MSC (dMSC) and differentiated ADSC (dADSC) in vitro.dMSC and dADSC were identified by p75 protein and gl ial fibrillary acidic protein (GFAP). SCs were isolated and culturedfrom 10 3-day-old SD mice with weighing 6-8 g. CEANA were made from bilateral sciatic nerves of 20 adult Wistar mice with weighing 200-250 g. Forty adult SD mice were made the model of left sciatic nerve defect (15 mm) and divided into 5 groups (n=8 per group) according to CEANA with different sources of SCs: autografting (group A), acellular grafting with SCs (5 × 105) (group B), acellular grafting with dMSCs (5 × 105) (group C), acellular grafting with dADSCs (5 × 105) (group D), and acellular grafting alone (group E). Motor and sensory nerve recovery was assessed by Von Frey and tension of the triceps surae muscle testing 12 weeks after operation. Then wet weight recovery ratio of triceps surae muscles was measured and histomorphometric assessment of nerve grafts was evaluated. Results BMSCs and ADSCs did not express antigens CD34 and CD45, and expressed antigen CD90. BMSCs and ADSC were differentiated into similar morphous of SCs and confirmed by the detection of SCs-specific cellsurface markers. The mean 50% withdrawal threshold in groups A, B, C, D, and E was (13.8 ± 2.3), (15.4 ± 6.5), (16.9 ± 5.3), (16.3 ± 3.5), and (20.0 ± 5.3) g, showing significant difference between group A and group E (P lt; 0.01). The recovery of tension of the triceps surae muscle in groups A, B, C, D, and E was 87.0% ± 9.7%, 70.0% ± 6.6%, 69.0% ± 6.7%, 65.0% ± 9.8%, and 45.0%± 12.1%, showing significant differences between groups A, B, C, D, and group E (P lt; 0.05). No inflammatory reactionexisted around nerve graft. The histological observation indicated that the number of myel inated nerve fiber and the myel in sheath thickness in group E were significantly smaller than that in groups B, C, and D (P lt; 0.01). The fiber diameter of group B was significantly bigger than that of groups C and D (P lt; 0.05) Conclusion CEANA supplementing with dADSC has similar repair effect in peripheral nerve defect to supplementing with dMSC or SCs. dADSC, as an ideal seeding cell in nerve tissue engineering, can be benefit for treatment of peripheral nerve injuries.