Objective?To review the recent progress of the researches in construction of tissue engineered osteochondral composites, and to discuss the challenges in construction of tissue engineered osteochondral composites.?Methods?The recent literature on the construction of tissue engineered osteochondral composites was extensively reviewed and analyzed.?Results?The studies on the construction of tissue engineered osteochondral composites are relatively more in vivo, the current focus is that different tissues derived mesenchymal stem cells are widely used to be seed cells; single-phase scaffold has been limited, studies on biphase scaffold and triphase scaffold are new trends; the design and performance of bioreactor need to be further optimized in the future.?Conclusion?The construction of tissue engineered osteochondral composites will be a promising method for the treatment of cartilage defects.
Objective To investigate the cellular compatibil ity of polyvinyl alcohol (PVA)/wild antheraea pernyisilk fibroin (WSF), and to explore the feasibil ity for tendon tissue engineering scaffold in vitro. Methods The solutions of WSF (11%), PVA (11%), and PVA/WSF (11%) were prepared with 98% formic acid (mass fraction) at a mass ratio of 9 : 1. The electrospinning membranes of WSF, PVA, and PVA/WSF were prepared by electrostatic spinning apparatus. The morphologies of scaffolds were evaluated using scanning electronic microscope (SEM). The tendon cells were isolated from tail tendon of 3-dayold Sprague Dawley rats in vitro. The experiment was performed using the 3rd generation cells. The tendon cells (1 × 106/mL) were cocultured with PVA and PVA/WSF electrospinning film, respectively, and MTT test was used to assess the cell adhesion rate 4, 12 hours after coculture. The tendon cells were cultured in PVA and PVA/WSF extraction medium of different concentration (1, 1/2, and 1/4), respectively; and the absorbance (A) values were detected at 1, 3, 5, and 7 days to evaluate the cytotoxicity. The composite of tendon cells and the PVA or PVA/WSF scaffold were observed by HE staining at 7 days and characterized by SEM at 1,3, 5, and 7 days. Results The solution of WSF could not be used to electrospin; and the solution of PVA and PVA/WSF could be electrospun. After coculture of tendon and PVA or PVA/WSF electrospinning membranes, the cell adhesion rates were 26.9% ±0.4% and 87.0% ± 1.0%, respectively for 4 hours, showing significant difference (t=100.400, P=0.000); the cell adhesion rates were 35.2% ± 0.6% and 110.0% ± 1.7%, respectively for 12 hours, showing significant difference (t=42.500, P=0.000). The cytotoxicity of PVA/WSF was less significantly than that of PVA (P lt; 0.05) and significant difference was observed between 1/2 PVA and 1/4PVA (P lt; 0.05). HE staining and SEM images showed that the tendon cells could adhere to PVA and PVA/WSF scaffolds, but that the cells grew better in PVA/WSF scaffold than in PVA scaffold in vitro. Conclusion PVA/WSF electrospinning membrane scaffold has good cell compatibility, and it is expected to be an ideal scaffold of tendon tissue engineering.
Objective To review the recent progress of the researches in the field of cartilage tissue engineering, and to discuss the challenges in construction of tissue engineered cartilage. Methods Literature related with cartilage tissue engineering was reviewed and analyzed. Results Some techniques have been appl ied in cl inical. As far as the seeding cells, induced pluripotent stem cells have attracted much more attention. Current strategies of scaffold designing are trying to imitate both component and structure of natural extracellular matrix. Cartilage regeneration through the autologous cell homing technique el iminate the transplantation of exotic cells and has become the hot topic. Conclusion Successful treatment of the damaged cartilage using tissue engineering method will depend on the advances of stem cell technology development, biomimetic scaffolds fabrication and proper appl ication of growth factors.
【Abstract】 Objective To develop a novel cartilage acellular matrix (CACM) scaffold and to investigate its performance for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and gradient centrifugation and made into 3% suspension after acellularization treatment. After placing the suspension into moulds, 3-D porous CACM scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-l inked by exposure to ultraviolet radiation and immersion in a carbodiimide solution 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysucinimide. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorption rate analysis. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by conditioned medium including TGF-β1, canine BMSCs were seeded into the scaffold. Cell prol iferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed that there are no chondrocytefragments in the scaffolds and that toluidine blue, safranin O and anti-collagen II immunohistochemistry staining werepositive. The novel 3-D porous CACM scaffold had good pore interconnectivity with pore diameter (155 ± 34) μm, 91.3% ± 2.0% porosity and 2 451% ± 155% water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs indicated that cells covered the scaffolds uniformly and majority of the cells showed the round or ell iptic morphology with much matrix secretion. Conclusion The 3-D porous CACM scaffold reserved most of extracellular matrix after thoroughly decellularization, has good pore diameter and porosity, non-toxicity and good biocompatibil ity, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.
Objective To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold in vitro. Methods BMSCs were obtained and isolated from 20 New Zealand white rabbits (5-6 months old). The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope (SEM). A full-thickness cartilage defect (6 mm in diameter) was established and 5 microholes (1 mm in diameter and 3 mm in depth) were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A (n=10) and B (n=10): culture of the marrow clot alone (group A) and culture of the marrow clot with transforming growth factor β3 (TGF-β3) (group B). Twenty rabbits which were punctured were randomly divided into groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clot composite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-β3 (group D). The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture. Results Cells were successfully induced into osteoblasts, chondrocytes, and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks (P lt; 0.05); group D was significantly larger than group C in size (P lt; 0.05). Few cells were seen, and no glycosaminoglycan (GAG) and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks (P lt; 0.05). Conclusion The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-β3-induced chondrogenic differentiation in vitro.
ObjectiveTo prepare human acellular adipose tissue matrix and to evaluate the cellular compatibility so as to explore a suitable bio-derived scaffold for adipose tissue engineering. MethodsThe adipose tissue was harvested from abdominal skin graft of breast cancer patients undergoing radical mastectomy or modified radical mastectomy, and then was treated with a series of decellularization processes including repeated freeze-thaw, enzyme digestion, and organic solvent extraction. The matrix was examined by histology, immunohistochemistry, DAPI fluorescence staining, and scanning electron microscopy to observe the the removal of cells and to analyze its composition of collagen type IV, laminin, and fibronectin, and microstructure. The 3rd passage human adipose-derived stem cells (hADSCs) were co-cultured with acellular adipose tissue matrix and different concentrations of extracted liquid (100%, 75%, 50%, and 25%). The cytotoxic effects of the matrix were tested by MTT. The biocompatibility of the matrix was detected by live/dead staining and scanning electron microscopy observation. ResultsThe acellular adipose tissue matrix basically maintains intrinsical morphology. The matrix after acellular treatment consisted of extracellular matrix without any cell components, but there were abundant collagen type I; neither DNA nor lipid residual was detected. Moreover, the collagen was the main component of the matrix which was rich in laminin and fibronectin. At 1, 3, and 5 days after co-cultured with hADSCs, the cytotoxic effect of matrix was grade 0-1. The matrix displayed good cell compatibility and proliferation. ConclusionThe acellular adipose tissue matrix prepared by repeated freeze-thaw, enzyme digestion, and organic solvent extraction method remains abundant extracellular matrix and has good cellular compatibility, so it is expected to be an ideal bio-derived scaffold for adipose tissue engineering.
ObjectiveTo observe the bladder regeneration by collagen membrane scaffolds for bladder construction to find a new alternative scaffold material. MethodsTwelve healthy adult male Sprague Dawley rats, weighing 300-350 g, were randomly divided into collagen membrane scaffold group (experimental group, n=6), and sham operated group (control group, n=6). Upper hemicystectomy was performed and collagen scaffold was used for reconstruction in experimental group, while the bladder was turned over without bladder resection in control group. At 30 days after operation, the animals were sacrificed and grafts were harvested;HE staining and Masson staining were used to evaluate the bladder regeneration, immunohistochemical staining was performed with α-smooth muscleactin (α-SMA) and von Willebrand factor (vWF) markers to evaluate the percentage of α-SMA positive area and capillary number. ResultsThe rats of 2 groups survived to the end of the experiment, and no urine leakage or infection was observed in experimental group. Histologically, control group presented a pattern of normal bladder structure, experimental group presented a pattern of almost normal urothelium with a small amount of smooth muscle cells and a thin layer of undegraded collagen fibers. Immunohistochemically, experimental group showed ingrowth of smooth muscle fibers and new capillary formation along the collagen membrane scaffolds. The percentage of α-SMA positive area and capillary number in experimental group were significantly lower than those in control group (6.49%±2.14% vs. 52.42%±1.78% and 4.83±0.75 vs. 14.83±1.17, respectively)(t=40.40, P=0.00; t=17.62, P=0.00). ConclusionThe collagen membrane scaffolds could be an effective scaffold material for bladder reconstruction.
Objective To study the preparation method of acellular vascular matrix and to evaluate its biocompatibil ity and safety so as to afford an ideal scaffold for tissue engineered blood vessel. Methods Fresh caprine carotids (length, 50 mm) were harvested and treated with repeated frozen (—80 )/thawing (37℃), cold isostatic pressing (506 MPa, 4 ), and 0.125% sodium dodecyl sulfate separately for preparation of acellular vascular matrix. Fluorescence staining and DNA remain test were used to assess the cell extracting results. Biological characteristics were compared with the raw caprine carotids using HE staining, Masson staining, scanning electron microscope (SEM), and mechanical test. Biocompatibil ity wasdetected using cell adhesion test, MTT assay, and subcutaneously embedding test. Ten SD rats were divided into 2 groups (n=5). In experimental group, acellular vascular matrix preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment and chemical detergent was subcutaneously embedded; and in control group, acellular vascular matrix preserved only by repeated frozen/thawing and ultrahigh pressure treatment was subcutaneously embedded. Results HE staining and Masson staining revealed that no nucleus was detected in the acellular vascular matrix. SEM demonstrated that a lot of collagen fibers were preserved which were beneficial for cell adhesion. Fluorescence staining and DNA remain test showed that the cells were removed completely. There was no significant difference in stress and strain under the maximum load between before and after treatment. Mechanical test revealed that the acellular vascular matrix reserved mechanical properties of the raw caprine carotids. Cell adhesion test and MTT assay confirmed that cytotoxicity was grade 0-1, and the acellular vascular matrix had good compatibil ity to endothel ial cells. After subcutaneously embedding for 8 weeks, negl igible lymphocyte infiltration was observed in experimental group but obvious lymphocyte infiltration in control group. Conclusion The acellular vascular matrix, which is well-preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment, and chemical detergent, is an ideal scaffold for tissue engineered blood vessel.
The engineered heart tissues (EHTs) present a promising alternative to current materials for native myocardial tissue due to the unique characteristics. However, until now, the clinical application of EHTs is limited by a serial of practical problems yet. Generally, the challenges need to further optimize include biomaterials, cell sources, and strategies of revascularization or establishment of EHTs. This review focuses on the newly progress on these aspects to encourage the emergence of novel EHTs that can meet clinic requirement properly.
ObjectiveTo summarize the research progress of several three-dimensional (3-D) printing scaffold materials in bone tissue engineering. MethodThe recent domestic and international articles about 3-D printing scaffold materials were reviewed and summarized. ResultsCompared with conventional manufacturing methods, 3-D printing has distinctive advantages, such as enhancing the controllability of the structure and increasing the productivity. In addition to the traditional metal and ceramic scaffolds, 3-D printing scaffolds carrying seeding cells and tissue factors as well as scaffolds filling particular drugs for special need have been paid more and more attention. ConclusionsThe development of 3-D printing porous scaffolds have revealed new perspectives in bone repairing. But it is still at the initial stage, more basic and clinical researches are still needed.