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Due to good mechanical properties and biocompatibility, tissue engineering scaffolds have become the vital method for repairing and regenerating articular cartilage defects. With the continuous development of tissue engineering technology, many scaffolds preparation and formation methods have been developed and tested in the past decade, however, the preparation of ideal regenerative scaffolds remain controversial. As load-bearing tissue inside the body joints, the matrix structure and cell composition of articular cartilage are hierarchical, and there are several smooth natural gradients from the cartilage surface to the subchondral bone layer, including cell phenotype and number, specific growth factors, matrix composition, fiber arrangement, mechanical properties, nutrient and oxygen consumption. Therefore, in the design of regenerative scaffolds, it is necessary to achieve these gradients to regenerate articular cartilage in situ. In recent studies, many new biomimetic gradient scaffolds have been used to simulate the natural gradient of articular cartilage. These scaffolds show different mechanical, physicochemical or biological gradients in the structure, and have achieved good repair effects. The related articles on tissue engineering for the treatment of articular cartilage defects were retrieved by searching databases with key wordsarticular cartilage injury, cartilage repair and gradient scaffolds. In this work,the structural, biochemical, biomechanical and nutrient metabolism gradients of natural articular cartilage were studied and summarized firstly. Then, the latest design and construction of articular cartilage gradient scaffolds were classified. Besides that, the material composition (such as hydrogels, nanomaterials, etc.) and the preparation process (such as electrospinning, 3D printing, etc.) of grandient scaffolds were further enhanced. Finally, the prospect and challenge of biomimetic gradient scaffolds in cartilage engineering are discussed, which provides a theoretical basis for the successful application of gradient scaffolds in clinical transformation.
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The treatment of articular cartilage (AC) injury caused by various reasons is still a major clinical problem. The emergence of cartilage tissue engineering brings new hope for the treatment of AC injury. In general, AC tissue engineering can be divided into two categories, including cell-based tissue engineering and cell-free tissue engineering. Although cell-based tissue engineering can repair cartilage damage to a certain extent, existing therapeutic strategies still suffer from limited cell sources, high costs, risk of disease transmission, and complex procedures. However, the cell-free tissue engineering avoids these shortcomings and brings hope for in-situ AC regeneration. Non-cellular tissue engineering is mainly used to recruit endogenous stem cells/progenitor cells (SCPCs) to reach the site of cartilage injury, and provide a suitable regenerative microenvironment to promote cell proliferation and chondrogenic differentiation, then the maturation of new cartilage tissue was promoted. Therefore, it is also called as cell-homing in situ tissue engineering. Successful recruitment of endogenous SCPCs is the first step in in-situ cartilage tissue engineering. This review aims to introduce chemokine response of cartilage injury, systematically summarize traditional chemoattractant (chemokines and growth factors etc.) and emerging chemoattractant (functional peptides, exosomes and nucleic acid adapters etc.), evaluate the combination mode between chemoattractant and delivery devices, discuss the prospects and challenges of chemoattractant-mediated in situ tissue engineering and provide theoretical basis for the design of endogenous SCPCs homing-based in situ tissue engineering.
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Objective: To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide (mHSP/P) vaccine in combination with a programmed death ligand 1 (PD-L1) inhibitor in mouse sarcoma. Methods: Immunohistochemical staining and en-zyme-linked immunosorbent assay (Elisa) was used to quantitatively identify the expression of heat shock proteins (HSP70, HSP90, Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide (Grp94/P) sarcoma vac-cines were isolated using chromatography and were identified by Western blot (WB). Flow cytometry was used to determine their cy-totoxic effects. The levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors. Results: Tumor tissue carries a variety of HSP subtypes (HSP70, HSP90, Grp94). We successfully isolated sarco-ma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cy-totoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/P stimulation (P<0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor (P<0.05). Conclusions: Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune eva-sion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.
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<p><b>BACKGROUND</b>To develop a vaccine-based immunotherapy for sarcoma, we evaluated a mixture of heat shock proteins (mHSPs) as a vaccine for sarcoma treatment in a mouse model. Heat shock protein/peptides (HSP/Ps) are autoimmune factors that can induce both adaptive and innate immune responses; HSP/Ps isolated from tumors can induce antitumor immune activity when used as vaccines.</p><p><b>METHODS</b>In this study, we evaluated the effects of mHSP/Ps on prophylactic antitumor immunity. We extracted mHSP/Ps, including HSP60, HSP70, GP96, and HSP110, from the mouse sarcoma cell lines S180 and MCA207 using chromatography. The immunity induced by mHSP/Ps was assessed using flow cytometry, ELISPOT, lactate dehydrogenase release, and enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>Of S180 sarcoma-bearing mice immunized with mHSP/Ps isolated from S180 cells, 41.2% showed tumor regression and long-term survival, with a tumor growth inhibition rate of 82.3% at 30 days. Of MCA207 sarcoma-bearing mice immunized with mHSP/Ps isolated from MCA207 cells, 50% showed tumor regression and long-term survival with a tumor growth inhibition rate of 79.3%. All control mice died within 40 days. The proportions of natural killer cells, CD8+, and interferon-γ-secreting cells and tumor-specific cytotoxic T-lymphocyte activity were increased in the immunized group.</p><p><b>CONCLUSIONS</b>Vaccination with a polyvalent mHSP/P cancer vaccine can induce an immunological response and a marked antitumor response to autologous tumors. This mHSP/P vaccine exerted greater antitumor effects than did HSP70, HSP60, or tumor lysates alone.</p>
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Animals , Female , Mice , Cancer Vaccines , Therapeutic Uses , Heat-Shock Proteins , Immunotherapy , Methods , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptides , Sarcoma, Experimental , VaccinationABSTRACT
Objective To probe the immunological traits of mesenchymal stem cells derived from umbilical cord Wharton's jelly (WJMSCs). Methods The diced Wharton's jelly which was from healthy fullterm birth human umbilical cord was cultured. The mesenchymal stem cells were identified with mesenchymal stem cells markers expression by flow cytometry and multiple differentiation ability. The expression of MHC- Ⅰ / Ⅱ, costimulatory molecules (CD40, CD80 and CD86) was detected with flow cytomctry, immunocytochemistry, and RT-PCR. The expression of immune inhibitors like HLA-G, IDO, and PGE2 was detected by immunocytochemistry and RT-PCR. The expression of immune-related molecules as IL-10, TGF-β, FGF and VEGF was detected with antibody microarray and western blot. Further more, to clarify the in vivo immune reaction of hWJMSCs, we fabricated the hWJMSC-scaffold constructs and implanted them into the rabbit backs. The lymphocyte infiltration and implanted cell survival observed with immunofluorescence. Results After culturinge of diced Wharton's jelly tissue, we obtained spindle-shaped cells. With differentiation medium, the cells can differentiate into osteoblasts, chongdrocytes, adipose cells and schwann cells. Expression of MHC, costimulatory molecules, and a series of immune suppressive-related molecules was found. Immune inhibitors as HLA-G, 1DO, PGE2, and immune suppressive related molecules as HGF, VEGF, TGFand IL-10 were positively expressed. But the cells did not express MHC-Ⅱ. No immune rejection was observed in vivo after implantation of hWJMSC-scaffold constructs. Conclusion It can be concluded that hWJMSCs have very low immunogenicity, which means the cells have potential to induce immune tolerance.The hWJMSCs do not provoke immune rejection in vivo.
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Objective To explore the effect of hepatocyte growth factor on peripheral nerve regeneration. Methods Sciatic nerve contusion injury was made by a custom-made clamp in Wistar rats,in which human hepatocyte growth factor expressed by adenoviral vector(Ad-HGF)was injected into the muscle around the injured nerve.The results of nerve regeneration were evaluated by sciatic nerve function index(SFI),muscle wet weight,neural electrophysiology and image analysis. Results Four weeks after sciatic nerve injury,the results of sciatic nerve function index(SFI),muscle wet weight,neural electrophysiology and image analysis showed better nerve regeneration in group injected with HGF than control group(P<0.05). Conclusion Hepatocyte growth factor can promote axon regeneration and functional recovery and is an effective neurotrophic factor for peripheral nerve regeneration after injury.
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@#Objective To investigate the antitumor immunity induced by tumor derived mixed heat shock protein/peptide(mHSPs),interleukin-12(IL-12)and Cyclophosphamide(Cy).MethodsPurified mixed HSP was prepared from tumor by S180 protein extraction and purification,SDS-PAGE,Western blot and animal experiment were applied for mixed HSPs analysis.ResultsThe proliferation of cytotoxic T lymphocyte(CTL)cocultured in the mHSPs+Cy+IL-12 group was significantly remarkable and the content of CD8+ CTLs was significantly more in comparison with the other groups(P<0.01).To the tumor bearing mice,mHSPs+Cy+IL-12 group showed partial therapeutic efficacy,the averaged survival period was over 60 d,and 90% of the mice in this group got long period tumor free survival(>90d),obvious difference(P<0.05)from the other groups.ConclusionTumor derived mixed HSPs can induce powerful antitumor immune efficacy and show favorable therapeutic efficacy.
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OBJECTIVE To observe the effect of electrolyzed oxidizing water(EOW) on the surface sterilization of indoor environment.METHODS Sterilized the surface of ground,wall,table,mob and thermostat-controlled water-bath in cell culture room and good laboratory practice(GLP) with EOW,then to draw the materials and culture with culture dish for 48 h in a 37℃ incubator,and count the colony number.Sterilization method and grouping: group A treated as a control,group B sterilized with EOW,group C sterilized with ultraviolate ray for 30 min and group D first treated with ultraviolate ray for 30 min,then sterilized with EOW.RESULTS In group A,the bacteria were overgrew and formed flakiness in 10/10 culture dishes;1 colony was formed in group B,the sterilization effective rate was 90%.The bacteria culture of group C found no bacteria growing after sterilization with ultraviolate ray,however,sample from surface and culture after sterilization were seen bacteria,though the number of bacteria was less than group A.The bacteria culture outcome of group D was negative.CONCLUSIONS The EOW has a good sterilization effect,it is safe and untoxic,costly cheap and convenient to use,and fit to claim of environmental protection.
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[Objective]To establish a method for obtaining well-differentiated goat chondrocytes quickly in large scale by RCSS using microcarriers technique. [Method]Articular chondrocytes were harvested from goat by sequential digestion with trypsin and collagenase , and then grown in RCSS bioreactor culture system which contained the Cytodex-3 microcarriers in the culture medium (DMEM) . Growth of chondrocytes on Cytodex-3 microcarriers was observed dynamically under phase contrast microscope . Immunocytochemical analysis was performed for type Ⅰcollagen and type Ⅱcollagen. [Result]The articular chondrocytes attached rapidly to the surface of Cytodex-3 microcarriers. Quick growth of these cells was observed after they fully spread onto the microcarriers. The density of chondrocytes increased by 15～17 times in later stage of culture as compared with the initial density. The harvested chondrocytes had no detectable staining for collagen type Ⅰ, but stained intensively for collagen type Ⅱ. [Conclusion]Microcarrier culture of chondrocytes can yield a large quantity of goat cells within a short time ,which will be of benefit for banking cartilage cells for reconstruction of impaired cartilage by way of tissue engineering.
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<p><b>OBJECTIVE</b>To investigate the relationship between immunogenicity and decellularization processes of chemically acellular nerve allografts.</p><p><b>METHODS</b>Adult Sprague Dawley rats were used as nerve donors and adult male Wistar rats used as nerve recipient hosts. 25 mm nerve segments were excised from SD rats' sciatic nerves. The nerve segments were decellularized via an improved chemical decelluarization treatment as follows: (1) nerve segments were rinsed with cold sterile Ringer's solution; (2) stabilized by pinning the ends to a thin plastic support, and submerged in 4% Triton-100 solution 12 h; (3) soaked into 3% sodium deoxycholate for 12 h; (4) washed in distilled water for 6 h. The procedures were repeated once again. The acellular nerve allografts from SD rats were sterilized by gamma irradiation and implanted into Wistar rats subcutanously. The control group was implantation of fresh nerve allografts from SD rats. The immunogenicity of acellular nerve allograft was tested by immunohistochemical examination of the intensity of CD3(+), CD4(+) and CD8(+) cells that infiltrated the allografts. Ulnar nerve segments were obtained from forearms of dogs and decellularized according to above procedures. According as the decellularization times, The ulnar nerve segments were divided into three subgroups: in group I, group II and group III, the nerve segments were decellularized repeatedly two, three and four cycles respectively. Each ulnar nerve segment was subdivided into five portions from proximal to distal end. The degrees of decellularization, demyelination and basal lamina integrity of extracellular matrix scaffold were observed with microscope and assessed by a score system. The immunohistochemical staining of GAG was observed.</p><p><b>RESULTS</b>The intensity of CD3(+), CD4(+) and CD8(+) T cells that infiltrated the allografts was greatly lower in acellular nerves than in fresh nerves. The mild cell-mediated host-graft immunorejection in acellular nerves was observed. On the decellularization procedures, the cells were completely extracted from nerves in all groups, but the myelin sheath were partially existed, and the GAG was present in the basal membrane of myelin sheath. In the score of demyelination, there were no statistical differences between groups (P > 0.05). The statistical difference of basal lamina integrity scores between group I and group II, group I and group III were significant (P < 0.05). As increasing the times of process, the degrees of disintegrity of basal lamina was significantly enhanced.</p><p><b>CONCLUSIONS</b>Although decellularization processes significantly reduce the cell-mediated immunorejection of acellular nerve allografts, it can induce mild immunoreaction all the same, the antigen that responsible for immunogenicity may be the residual component of GAG in myelin sheath.</p>