Entity

Time filter

Source Type


Xu H.,Cell Engineering Laboratory of Orthopaedic Institute
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery | Year: 2013

To fabricate a novel composite scaffold with acellular demineralized bone matrix/acellular nucleus pulposus matrix and to verify the feasibility of using it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties. Pig proximal femoral cancellous bone rings (10 mm in external diameter, 5 mm in internal diameter, and 3 mm in thickness) were fabricated, and were dealed with degreasing, decalcification, and decellularization to prepare the annulus fibrosus phase of scaffold. Nucleus pulposus was taken from pig tails, decellularized with Triton X-100 and deoxycholic acid, crushed and centrifugalized to prepare nucleus pulposus extracellular mtrtix which was injected into the center of annulus fibrosus phase. Then the composite scaffold was freeze-dryed, cross-linked with ultraviolet radiation/carbodiimide and disinfected for use. The scaffold was investigated by general observation, HE staining, and scanning electron microscopy, as well as porosity measurement, water absorption rate, and compressive elastic modulus. Adipose-derived stem cells (ADSCs) were cultured with different concentrations of scaffold extract (25%, 50%, and 100%) to assess cytotoxicity of the scaffold. The cell viability of ADSCs seeded on the scaffold was detected by Live/Dead staining. The scaffold was white by general observation. The HE staining revealed that there was no cell fragments on the scaffold, and the dye homogeneously distributed. The scanning electron microscopy showed that the pore of the annulus fibrosus phase interconnected and the pore size was uniform; acellular nucleus pulposus matrix microfilament interconnected forming a uniform network structure, and the junction of the scaffold was closely connected. The novel porous scaffold had a good pore interconnectivity with (343.00 +/- 88.25) microm pore diameter of the annulus fibrosus phase, 82.98% +/- 7.02% porosity and 621.53% +/- 53.31% water absorption rate. The biomechanical test showed that the compressive modulus of elasticity was (89.07 +/- 8.73) kPa. The MTT test indicated that scaffold extract had no influence on cell proliferation. Live/Dead staining showed that ADSCs had a good proliferation on the scaffold and there was no dead cell. Novel composite scaffold made of acellular demineralized bone matrix/acellular nucleus pulposus matrix has good pore diameter and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, and good biocompatibility, so it is a suitable scaffold for intervertebral disc tissue engineering. Source


Xu H.-W.,Tianjin Hospital | Xu B.-S.,Tianjin Hospital | Yang Q.,Tianjin Hospital | Li X.-L.,Cell Engineering Laboratory of Orthopaedic Institute | And 5 more authors.
Yiyong Shengwu Lixue/Journal of Medical Biomechanics | Year: 2013

Objective: To investigate effects of different decellularization methods on biomechanical properties and histological structure of annulus fibrosus in pigtails and provide experimental evidence for the construction of tissue engineering annulus fibrosus. Methods: Sixty fresh annulus fibrosus were dissected from caudal disks of pigs and randomly assigned to 4 groups with 15 in each group. Triton X-100 group(Group A): annulus fibrosus were treated with hypotonic Tris-HCl buffer for 48 hours and de-cellularized with Triton X-100, DNase I and RNase A. SDS group (Group B): annulus fibrosus were subjected to 3 cycles of freeze-thaw and subsequently de-cellularized with SDS, DNaseI and RNase A. Trypsin group (Group C): annulus fibrosus were de-cellularized with Tris buffer containing trypsin, DNase I and RNase A. Control group: fresh annulus fibrosus underwent no treatment. After the decellularization process was completed, hematoxylin-eosin (HE) staining was conducted to examine the efficacy on cell removal, and the ultrastructure of annulus fibrosus were observed by scanning electron microscopy. The collagen content, glycosaminoglycan (GAG) content and biomechanical parameters in each group were also detected. Results: HE staining and scanning electron microscopy showed that no residual cells were found in Group A, B and C. The structure of annulus fibrosus in Group A was not disturbed, while that in Group B and C was damaged severely and slightly, respectively. There was no statistical difference in collagen content among Group A, B and C, as compared to the control group (P>0.05). But the GAG content was significantly more lower in Group A, B and C than in the control group (P<0.05). There was no statistical difference in ultimate load, ultimate stress, toughness, elastic modulus and mechanical work to fracture between Group A, C and control group (P>0.05), while these parameters of Group B were lower than those in the control group (P<0.05). Conclusions: The Triton X-100-treated annulus fibrosus retained the major extracellular matrix composition after cell removal and preserved the major structure and mechanical strength, which is preferable for the construction of tissue engineering annulus fibrosus. Source


Xu H.,Tianjin Hospital | Xu H.,Tianjin Medical University | Xu B.,Tianjin Hospital | Yang Q.,Tianjin Hospital | And 9 more authors.
PLoS ONE | Year: 2014

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. © 2014 Xu et al. Source


Xu B.,Tianjin Hospital | Xu H.,Tianjin Hospital | Xu H.,Tianjin Medical University | Wu Y.,Tianjin Hospital | And 6 more authors.
PLoS ONE | Year: 2015

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. © 2015 Xu et al. Source


Zhao B.,Tianjin Medical University | Ma X.-L.,Tianjin Medical University | Ma X.-L.,Cell Engineering Laboratory of Orthopaedic Institute | Sun X.-L.,Cell Engineering Laboratory of Orthopaedic Institute | And 6 more authors.
Yiyong Shengwu Lixue/Journal of Medical Biomechanics | Year: 2013

Objective: To study the morphology and biomechanical properties of the improved acellularized nerve scaffold using the technique of hypotonic buffer combined with freeze-drying. Methods: The traditional acellularized nerve scaffold (traditional group) was made to be improved with the technique of hypotonic buffer combined with freeze-drying (improved group). After the acellularization process was completed, the histological structure of nerves in each group was observed by HE staining and scanning electron microscope. The interval porosity and void diameter in each group were measured by Mimics software. The biomechanical properties of nerves in each group were tested by mechanical apparatus (Endura TEC ELF3200). Results: The acellularization effect of the improved chemical method with the technique of hypotonic buffer combined with freeze-drying was similar to that of the traditional Hudson method, but the histological structure was more porous in improved group than that in traditional group. The interval porosity of traditional group and improved group were 34.5% and 49.3%, respectively; the void diameter of traditional group and improved group were 11.96 and 17.61 μm, respectively. Biomechanical testing results showed that there was no statistical difference in ultimate load, ultimate stress, ultimate strain and mechanical work to fracture in each group (P>0.05). Conclusions: The acellularized nerve prepared by hypotonic buffer combined with freeze-drying can be used as a new kind of nerve scaffold material to make better contribution to cell combination. Source

Discover hidden collaborations