Comparison of intensity-modulated radiation therapy alone vs. intensity-modulated radiation therapy combined with chemotherapy in elderly nasopharyngeal carcinoma patients (aged >65 years).

PURPOSE: The efficacy and tolerability of adding chemotherapy to radiotherapy in the era of intensity-modulated radiation therapy (IMRT) remain controversial among older patients with nasopharyngeal carcinoma (NPC). The present study compared IMRT alone with IMRT in combination with chemotherapy in elderly NPC patients. METHODS: Between January 2011 and December 2014, 102 patients aged >65 years with NPC who received IMRT alone (IMRT group) or IMRT in combination with chemotherapy (IMRT/CT group) were enrolled. Patients from both treatment arms were pair-matched (1:1 ratio) based on six clinical factors. Differences in overall survival (OS), disease-free survival (DFS), locoregional relapse-free survival (LRRFS), and distant metastasis-free survival (DMFS) were assessed using the Kaplan-Meier method and Cox proportional hazards models, whereas the toxicity profile was assessed using Common Terminology Criteria for Adverse Events (CTCAE) version 4. RESULTS: No significant differences were noted in OS (72.1% vs. 72.5%, p = 0.799), DFS (65.9% vs. 70.1%, p = 0.733), LRRFS (76.4% vs. 71.6%, p = 0.184), and DMFS (90.8% vs. 98.0%, p = 0.610) between the IMRT and IMRT/CT groups. Multivariate analyses showed that chemotherapy was not an independent factor for OS, DFS, LRRFS, and DMFS. However, the incidences of grade 3 vomiting/nausea (p = 0.000), leukopenia/neutropenia (p = 0.000), thrombocytopenia (p = 0.041), and anemia (p = 0.040) were significantly higher in the IMRT/CT group compared with the IMRT group. No grade 4 toxicities were observed. CONCLUSION: IMRT alone was similar to IMRT/CT in treating elderly NPC patients (age >65 years), with comparable survival outcomes and less grade 3 toxicities.

One-Year Relapse of Mandibular Distraction for Hemifacial Microsomia Using Masseteric Botulinum Toxin Type A Injections.

BACKGROUND: The relapse of hemifacial microsomia was thought to be highly related to the soft tissue envelope around the mandible angle mainly composed by masseter and medial pterygoid. According to the reason, we tried to apply masseter injection of type A botulinum toxin to weaken the soft envelope tension on the early stage post mandible distraction in adult HFM patients. METHODS: Eight patients diagnosed with HFM were studied and randomly assigned to an experimental or control group. Patients in the experimental group were treated with DO, orthognathic surgeries, autologous fat grafting, and bilateral masseter muscle injection with type A botulinum toxin. The patients in control group were treated with the same procedures as the patients in experimental group except for masseter muscle injection with type A botulinum toxin. The recurrence rates of both groups were evaluated and analyzed after nearly 1 year of follow-up. RESULTS: The mean recurrence rate was 26.30% ±â€Š11.84% (range 7.62%-37.27%) in the 8 patients after 1-year follow-up. The relapse rate was 16.32% ±â€Š7.78% (7.62%-26.22%) in the experimental group and 36.28% ±â€Š1.03% (34.84%-37.27%) in the control group. There was a significant difference (P = 0.002) between the experimental group and the control group. CONCLUSIONS: The combination of DO, orthognathic surgeries, autologous fat particle transplantation, and masseter muscle type A botulinum toxin injection technique could be a comprehensive treatment plan for adult patients of HFM. Furthermore, masseter injection of type A botulinum toxin might be an alternative method to reduce the early recurrence rate of postoperative adult patients of HFM.

Seasonal Variations in Microtia of Chinese Population.

BACKGROUND: Seasonality of congenital birth defect could help to identify environmental risk factors. Data concerning the seasonality of the prevalence of microtia are little. This article aims to determine whether births of microtia follow a certain pattern. METHODS: Data were obtained from 2669 patients with microtia who were admitted to Second Ear Reconstruction Center of Plastic Surgery Hospital, Chinese Academy of Medical Science from January 2007 to December 2013. The controls consist of all living births from the Obstetric Department of the Haidian Maternal & Child Health Hospital during the same time. Seasonal variations in months of births were analyzed by using χ test. RESULTS: A total of 2669 patients with microtia and 89,273 healthy living newborns were included in this study. Birth time peak of the patients occurred in autumn, especially in November, compared with the nadir in the spring, especially in April (P G 0.05). The birth time peak of male patients occurred in autumn, too, especially in October and November, While the valley occurred in spring (April, too). However, the seasonality in female patients is not so apparent with the peak occurred in the tail of summer and autumn, especially in August, November, and September orderly, while the valley occurred in March. CONCLUSIONS: There is a possible seasonality in birth months and a difference between sexes of patients with microtia in this native Chinese population. This approach could be useful to study the etiology of microtia.

Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs.

The Retrograde Transposition of the Remnant Earlobe in Patients With Low-Set Microtia.

During auricle reconstruction, lobular transposition has become a routine technique applied by most of surgeons. But to some low-set remnant ears, it is difficult to manipulate the conventional lobule transposition method in clinical application. In this article, the authors introduce a method to retrogradely transpose the remnant ear with the the ratio of length:width of the lobular flap being 4-5:1. The lobule transposition could be applied during the first stage of Nagata method or the third stage using expansion method. The authors take the superior part of the remnant ear as the pedicle and make the incision at the middle and inferior parts of the remnant ear to form the lobular flap. Then the inferior lobule is rotated posteriorly and superiorly to cover the rear end of the framework and to form the inferior part of helical rim. The results of the reconstructed auricles are satisfactory with aesthetic natural earlobes and the location of the reconstructed ear is symmetric to the contralateral ear. The authors believe that to the 2% to 5% patients with low-set microtia, this is a good way to make use of remnant ear for the purpose of a real earlobe.

Use of tissue engineering in treatment of the male genitourinary tract abnormalities.

INTRODUCTION: A variety of congenital and acquired male genitourinary tract abnormalities can lead to organ damage or tissue loss that requires surgical reconstruction. Traditional reconstructive methods do not produce consistent satisfactory structural or functional replacement and may damage the genitourinary tract. Tissue engineering provides a promising alternative for the treatment of these disorders. AIM: The aim of this article is to provide an update on clinical and experimental evidence concerning the application of tissue engineering to treatment of abnormalities in the male genitourinary tract system. METHODS: A PubMed search was performed to retrieve relevant clinical and basic literature. MAIN OUTCOME MEASURES: The topics discussed in this review include the experimental and clinical application of tissue engineering for reconstruction of the urethra, penis, testis, and prostate. RESULTS: Tissue engineering techniques can provide a plentiful source of healthy tissue for reconstructive purposes. Acellular matrix scaffold and seed cells are two key elements in tissue engineering. Proper employment of seed cells and scaffold material may result in synergistic effects. Moreover, new tissue engineering technologies are being transferred from the laboratory to clinical practice. CONCLUSIONS: Tissue engineering provides biological substitutes that can restore and maintain normal function in diseased and injured tissues, thus providing an effective technique for regeneration of the male genitourinary tract.

[Reconstruction of rabbit corneal stroma with skin fibroblasts].

OBJECTIVE: To explore whether skin fibroblasts could be used as a cell source for reconstruction of the corneal stroma. METHODS: It was an experimental study. Skin fibroblast cells were isolated from newborn rabbits, cultured and expanded in vitro. Cells were labeled with green fluorescence protein (GFP) gene by retro-viral infection. Fibroblasts at passage 3 were seeded on polyglycolic acid (PGA) non-woven fibers to form a cell-scaffold construct. Constructs were then implanted into the adult rabbit corneal stroma layer after being cultured in vitro for 1 week. Engineered stroma were observed continuously and harvested after 8 weeks of transplantation for gross, histological evaluation and Keratocan examination. PGA alone was used as control. RESULTS: The engineered tissue in the cornea became transparent gradually over a period of 8 weeks. Histological analysis showed that engineered stromal lamellar was relatively regular and the orientation of fibers was parallel to the surface of cornea, which is similar to normal cornea. The implanted cells were confirmed by GFP expression under fluorescent microscope, which also express Keratocan. By transmission electron microscopy examination, no significant difference in the diameter of collagen fiber was observed between engineered stroma (33.08 + or - 2.47) nm and normal stroma (t = 1.80, P = 0.0771). CONCLUSION: Skin fibroblast cells could be used as seed cells for reconstruction of the corneal stroma.

[In vitro tendon engineering using human dermal fibroblasts].

OBJECTIVE: To examine the feasibility of using human dermal fibroblasts (DFbs) and polyglycolic acids (PGA) to engineer tendon in vitro. METHODS: Human dermal fibroblasts (DFbs) were isolated from the foreskin tissues of children obtained during operation with collagenase and cultured in vitro. Human tendon was obtained from a patient undergoing amputation during operation to isolate tenocytes. The DFbs of second passage were seeded on PGA fibers to form cell-scaffold constructs in shape of tendons. Those constructs were divided into 4 groups: experimental group (n = 15) with the DFbs inoculated on PGA scaffold under constant tension generated by a U-shaped spring, control group 1 (n = 15) with the DFbs inoculated on PGA scaffold without tension, control group 2 (n = 3), i. e., cell-free pure PGA scaffolds under tension, and control group 3 (n = 5), i. e., tenocyte-scaffold constructs under tension that was harvested only at the ninth week. Samples were harvested 2, 5, 9, 14, and 18 weeks later to undergo histological examination and biomechanical test. RESULTS: Two weeks later histological examination showed that the constructs were mainly composed of PGA fibers in both the experimental group and the group without tension. Transmission electron microscopy showed fine cell attachment and stretching on the scaffold. By the 5th week, a neo-tendon was formed in all groups except for the cell-free group, and histology revealed the formation of collagen fibers. At the 9th week, the PGA fibers of the cell-free group were broken and partially degraded, the neo-tendon's diameter of the experimental group was (1.18 +/- 0.25) mm, significantly thinner than that of the group without tension[ (2.43 +/- 0.49) mm, P = 0.017]. The gross morphology of tendons of the experimental group and tenocyte group were similar to each other except for more cells in the experimental group. In experimental group, immunohistochemistry revealed the production of fibers of collagen type I & III that were aligned longitudinally along the force axis like the normal tendon pattern. An irregular collagen pattern was observed in the group without tension. The maximum tensile stress of the experimental group was (2.75 +/- 0.59) MPa, similar to that of the tenocyte group [(3.08 +/- 0.30) MPa, P = 0.439], and significantly greater than that of the group without tension [(0.82 +/- 0.21) MPa, P = 0.006]. At the 14th week the PGA fibers of the cell-free group were mostly degraded. In addition, more dead cells and tissue atrophy were observed in the experimental group, and the tensile stress was higher than that of the same group by the 9th week. In the 18th week the number of hollow fiber of the experimental group was more obvious, the number of dead cells increased, and the tensile stress was lower, however, there was no significant difference in other characteristics compared with those in the 14th week. CONCLUSIONS: DFbs can be used for in vitro tendon engineering as tenocytes. Mechanical stimulation by statistic strain is beneficial for tissue formation, but the effect may not be optimal if the tension is applied for too long.

[Construction of urethra mucosa structure in vitro by tissue engineering].

OBJECTIVE: To construct a structure of urethra mucosa in vitro by tissue engineering. METHODS: Primary porcine urothelial cells (UC) were obtained from the porcine bladder by enzymatic digestion and detected by immunofluorescence and RT-PCR. Bladder acellular matrix grafts (BAMG) were prepared, used as the scaffold and then evaluated by HE staining, Masson's trichrome staining, immunohistology and scanning electron microscopy. After in vitro culture and amplification, the UCs were seeded on the luminal surface the BAMGs. RESULTS: After 1 week of in vitro culture, the UCs formed a multilayer structure on the luminal surface of the BAMGs along the basement membrane. The tissue-engineered urothelium and BAMG complex was well formed and pan cytokeratins were positively expressed in the UCs on the scaffold. CONCLUSION: By tissue engineering, the urethra mucosa structure can be rapidly constructed in vitro, which can be applied to the repair of such urethral defect as hypospadias and urethral stricture.

[Androgen-secreting tissue construction with co-cultured rat testes somatic cells by tissue engineering technique].

OBJECTIVE: To explore the feasibility of constructing androgen-secreting tissue of a certain size and shape using co-cultured somatic cells of rat testis. METHODS: Thirty male Wistar rats were castrated. model and implanted rat model were prepared by resecting bilateral testes. The suspension of mixed testes cells was cultured to obtain various somatic cells of testes and Leydig cells were collected by differential anchorage-dependent method. These two kinds of cells were seeded onto biodegradable scaffolds of polyglycolic acid (PGA) fibers and cultured in vitro. The tissue formation of cell-scaffold constructs was observed by optical microscope and electronic microscope and the level of testosterone in the supernatant was detected regularly. After 7-day culture in vitro, the 2 kinds of cell-scaffold constructs, scaffold with purified Leydig cells or co-cultured testis somatic cells (seed cells), were implanted into the gastrocolic omentum or cavity of tunica vaginalis of the castrated rats. The implants were harvested 4, 6, 9, 12, and 24 weeks later to evaluate the tissue formation of cell-scaffold constructs in vivo. The serum testosterone level of the implanted rats was assayed to evaluate the testosterone secreting function of the regenerative tissue. RESULTS: Both the co-cultured testis somatic cells and Leydig cells had fine compatibility with the PGA fibers and adhered to the scaffolds very well. Testosterone was detected at a certain degree in the supernatant of cell-scaffold constructs, indicating the testosterone secreting function of the constructs. Two months after the implantation both kinds of cell-scaffold constructs formed testosterone secreting tissue in both gastrocolic omentum and cavity of tunica vaginalis of the implanted rats. The regenerative tissues were vascularized very well with a certain size and shape. Six weeks after implantation the serum testosterone level of the Leydig cell group was 0.60 ng/ml +/- 0.04 ng/ml, and that of the co-culture group was 0.84 ng/ml +/- 0.03 ng/ml, both significantly higher than that of the control castrated rats (0.56 ng/ml +/- 0.05 ng/ml, both P < 0.01), and the serum testosterone level of the co-cultured testes somatic cell implantation group was significantly higher than that of the Leydig cell implantation group too (P < 0.01). CONCLUSION: It is completely feasible to construct androgen-secreting tissue in vitro and in vivo using tissue engineering technique. Co-cultured testis somatic cells may serve as the better seed cells for androgen-secreting tissue engineering than purified Leydig cells in terms of the quantity and function of cells.

[Influence of transforming growth factor-beta1 inducing time on chondrogenesis of bone marrow stromal cells (BMSCs): in vitro experiment with porcine BMSCs].

OBJECTIVE: To explore the influence of transforming growth factor (TGF)-beta1 inducing time on the chondrogenesis of bone marrow stromal cells (BMSC), and on the construction of tissue engineering cartilage. METHODS: BMSCs were obtained from the greater trochanters of 3 pigs, cultured, seeded onto the cylindrical scaffolds made of polyglycolic acid at the density of 5.0 x 10(7) cells/ml, and then cultured with chondrogenesis media containing TGF-beta(1) (10 ng/ml), insulin-like growth factor-I (50 microg/L), and dexamethasone (40 microg/L) to be induced by TGF-beta(1) for 2 weeks (Group A), 4 weeks (Group B), 6 weeks (Group C), 8 weeks (Group D), or 10 weeks (Group E) respectively. 10 weeks later the cylindrical scaffolds underwent gross observation and histological examination. Alcin blue method was used to examine the content of proteoglycan (GAG). Immunochemistry and Western blotting were used to examine the type II collagen. The cylindrical scaffolds underwent biomechanical analysis. RESULTS: HE staining showed cartilage lacunae increasing in number from the periphery to center of the cylindrical scaffolds with the extended inducing time, and were arranged more uniformly progressively. Histochemical staining showed GAG accumulation. Immunohistochemistry and Western blotting showed that the content of type II collagen increased gradually, the amount of type II collagen stabilized after 6 weeks' culture, and there was no significant difference in the content of type II collagen between Group C and Group E. Biomechanical analysis showed that the modulus of elasticity and compression strength of the groups induced for more than 6 weeks (Groups C, D, and E) were all higher then those of Groups and B. CONCLUSION: TGF-beta(1) inducing time is correlated with the cartilage engineering characteristics with BMSC as seed cells. Induction for 6 weeks helps construct tissue engineered cartilage with good tissue structure, chemical composition and biomechanical properties.

[Potential of chondrogenesis of bone marrow stromal cells co-cultured with chondrocytes on biodegradable scaffold: in vivo experiment with pigs and mice].

OBJECTIVE: To explore the feasibility of in vivo chondrogenesis of bone marrow stromal cells (BMSCs) co-cultured with chondrocytes on biodegradable scaffold. METHODS: Porcine BMSCs were isolated, expanded and labeled with enhanced green fluorescent protein (EGFP), and then were mixed with articular chondrocytes isolated from porcine knee joint at the ratio of 1:1. The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml (co-culture group). Pure chondrocytes and BMSCs of the same ultimate concentration were seeded respectively onto the scaffold as positive control group and negative control group. After two weeks' culture in vitro, they were planted subcutaneously into nude mice respectively. These specimens were collected after in vivo implantation for 8 weeks to undergo microscopy. Laser confocal microscopy was used to observe the distribution of EGFP-labeled cells in the tissue. RT-PCR was used to examine the expression of collagen type II and aggrecan. Immunohistochemistry was used to observe the protein expression of collagen type II. RESULTS: The cell-scaffold constructs of the co-culture group and positive control group, could maintain the original size and shape no matter in vitro or in vivo. After 8 weeks' in vivo implantation, the constructs in both co-culture group and positive control group formed cartilage-like tissue with typical histological structure and extracellular matrix staining similar to those of the normal cartilage. The GAG content and compressive modulus of the co-culture group reached over 80% of those of the positive control group. Confocal microscopy revealed the presence of EGFP-labeled cells in the engineered cartilage lacuna. Histological examination showed that the constructs of the negative control group shrunk gradually after in vivo implantation with no typical cartilage-like tissue formation. CONCLUSION: In vitro co-cultured BMSC-chondrocyte-PGA constructs have the potential to form mature cartilage-like tissue in subcutaneous non-chondrogenesis environment, indicating that chondrocytes still provide enough signals for BMSC chondrogenic differentiation.

Cryopreservation of human embryonic stem cells: a protocol by programmed cooling.

Human embryonic stem (ES) cells have far-reaching applications in the areas of tissue engineering, regenerative medicine, pharmacology and basic scientific research. Although the culture conditions can maintain the human ES cells in an undifferentiated state for a transient period, spontaneous differentiation has also been observed during the routine culturing of ES cells. However, the maintenance of ES cells in the undifferentiated, pluripotent state for extended periods of time will be required in many areas of scientific research. Cryopreservation is a technology with potentially far reaching implication for the development and widespread use of such cell lines. This study was undertaken to develop and optimize a protocol for cryopreservation of human ES cells through programmed cooling. The effects of the seeding temperature, the cooling rate and the sub-zero temperature to which the samples were cooled before plunging into liquid nitrogen(the terminal temperature), all significantly affected the recovery of cryopreserved ES cells. After studying these factors, an improved protocol was obtained: the sample was cooled from 0 degree C to -35 degree C at a cooling rate of 0.5 degree per min, with seeding was set at -10 degree C, before being plunged immediately into the liquid nitrogen. Using this protocol, 9 of 11 colony fragments survived freezing and thawing and could be cultured for prolonged periods. They retained the properties of pluripotent cells, had a normal karyotype and showed histochemical staining for alkaline phosphatase.

Comparison of ultrasound biomicroscopy and spectral-domain anterior segment optical coherence tomography in evaluation of anterior segment after laser peripheral iridotomy.

AIM: To quantitatively assess narrow anterior chamber angle using spectral-domain anterior segment optical coherence tomography (SD-AS-OCT) and ultrasound biomicroscopy (UBM), and to evaluate the correlations and consistency between SD-AS-OCT and UBM. METHODS: Fifty-five eyes from 40 patients were examined. Patients were diagnosed with primary angle-closure glaucoma (PACG) remission (11 eyes from 8 patients), primary angle closure (PAC, 20 eyes from 20 patients) and PAC suspect (24 eyes from 12 patients). Each eye was examined by SD-AS-OCT and UBM after laser peripheral iridotomy (LPI). The measurements of SD-AS-OCT were angle open distance (AOD), anterior chamber angle (ACA), trabecular iris angle (TIA), and trabecular iris space area (TISA). UBM measurements were AOD and TIA. Correlations of AOD500 and TIA500 between UBM and AS-OCT were assessed. All parameters were analysed by SPSS 16.0 and MedCalc. RESULTS: ACA, TIA and AOD measured by SD-AS-OCT reached a maximum at the temporal quadrant and minimum at the nasal quadrant. TISA reached the maximum at the inferior and minimum at the superior quadrant. Group parameters of AOD500 and AOD750 showed a linear positive correlation, and AOD750 had less variability. UBM outcomes of AOD500 and TIA500 were significantly smaller than those of SD-AS-OCT. The results of the two techniques were correlated at the superior, nasal and inferior quadrants. CONCLUSION: Both UBM and SD-AS-OCT are efficient tools for follow-up during the course of PACG. We recommended using parameters at 750 µm anterior to the sclera spur for the screening and follow-up of PACG and PAC. The two methods might be alternatives to each other.

Use of autologous tissue engineered skin to treat porcine full-thickness skin defects.

OBJECTIVE: To explore a feasible method to repair full-thickness skin defects utilizing tissue engineered techniques. METHODS: The Changfeng hybrid swines were used and the skin specimens were cut from the posterior limb girdle region, from which the keratinocytes and fibroblasts were isolated and harvested by trypsin, EDTA, and type II collagenase. The cells were seeded in Petri dishes for primary culture. When the cells were in logarithmic growth phase, they were treated with trypsin to separate them from the floor of the tissue culture dishes. A biodegradable material, Pluronic F-127, was prefabricated and mixed with these cells, and then the cell-Pluronic compounds were seeded evenly into a polyglycolic acid (PGA). Then the constructs were replanted to the autologous animals to repair the full-thickness skin defects. Histology and immunohistochemistry of the neotissue were observed in 1, 2, 4, and 8 postoperative weeks. RESULTS: The cell-Pluronic F-127-PGA compounds repaired autologous full-thickness skin defects 1 week after implantation. Histologically, the tissue engineered skin was similar to the normal skin with stratified epidermis overlying a moderately thick collageneous dermis. Three of the structural proteins in the epidermal basement membrane zone, type IV collagen, laminin, and type VII collagen were detected using immunohistochemical methods. CONCLUSIONS: By studying the histology and immunohistochemistry of the neotissue, the bioengineered skin graft holds great promise for improving healing of the skin defects.

[Human adipose derived stem cells suppress lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli].

OBJECTIVE: To study immunological properties of adipose derived stem cells (ADSC) and their in vitro immunomodulatory effects on lymphocytes. METHODS: ADSC was isolated from fat tissue by liposuction and culture expanded. Cells at passage 2 were observed for the expression of HLAI, HLAII by FACs analysis. Peripheral blood lymphocytes (PBL) were cultured with allogeneic ADSCs at dose of 1 x 10(5). A third-party ADSCs at dose of 1 x 10(2), 1 x 10(3), 1 x 10(4), 1 x 10(5) were added to the ongoing two-way mixed lymphocyte reactions (MLR) for 6 days. Value of CPM (count per minute) was calculated with luminescence counter. RESULTS: Flow cytometry showed that undifferentiated ADSCs express HLA class I but not class II. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic ADSCs as stimulators. Results were similar using ADSCs pretreated with IFN-gamma. ADSCs elicited inhibit function of mixed lymphocyte cultures in a dose dependent way. Even if ADSCs were pretreated with IFN-gamma, the suppression ability was maintained. CONCLUSION: Undifferentiated ADSCs do not elicit alloreactive lymphocyte proliferative responses and could modulate immune responses in vitro.

[Cartilage-derived morphogenetic protein 1 initiates chondrogenic differentiation of human dermal fibroblasts in vitro].

OBJECTIVE: To explore the factors influencing the differentiation of fibroblasts into chondrocyte phenotype induced by a growth factor, cartilage-derived morphogenetic protein 1 (CDMP1). METHODS: Fibroblasts isolated from foreskin obtained during circumcision were cultured in the forms of micromass and monolayer culture. The culture fluid of the fibroblasts at the passage 2, 7, and 10 was added with CDMP1 of the concentrations at the concentrations of 10, 30, 100, and 300 ng/ml respectively and co-cultured for 7 days. RT-PCR was used to detect the expression of bone morphogenetic protein receptor (BMPR), activin receptor-like kinase (ALK) receptor, collagen types II, IV, and X before and after CDMP1 induction. Western blotting was used to detect the protein expression of collagen types II, a transcriptional factor Sox9, and aggrcan before and after the induction. Flow cytometry was used to detect the superficial markers CD29, CD105, CD106, and CD166, and the expression of collagen types I and II. RESULTS: Western blotting showed that the collagen type II positive cell rate in the passage 5 cells was 74.3% +/- 0.4%, not significantly different from that of the passage 2 cells (73.4% + 0.5%). When the concentrations of CDMP1 were 10 and 30 ng/ml no expression of aggrecan and collagen type II was detected, When the concentrations of CDMP1 was 100 and 300 ng/ml, the expression of aggrecan and collagen type II could be detected and without significant differences between these 2 concentrations. The expression of aggrcan and collagen type II mRNA disappeared in the monolayer cultured P2 and P5 fibroblasts induced by CDMP1 for 14 days, However, RT-PCR and Western blotting showed expression of collagen type II, aggrcan and SOX9 in the micromass cultured fibroblasts. RT-PCR showed that all fibroblasts cultured in vitro expressed ActR-I/ALK-2, BMPR-IA/ALK-3, and BMPR-IB/ALK-6 genes, and the expression of these genes significantly increased after CDMP1 for 7 days. CONCLUSION: CDMP1 stimulates the human dermal fibroblasts expanded in vitro to differentiate into chondrogenic phenotype in a dose dependent manner. Three-dimensional culture environments accelerate the chondrogenic differentiation. The expression of ALK receptors may involve the CDMP1 stimulated differentiation of fibroblasts.

[Stem cell, the basis for tissue and organ reconstruction].

Seed cells are prerequisite for reconstruction of artificial tissues/organs by tissue engineering approach. It has been widely accepted that stem cells are the best candidate for tissue engineering. The successful repair of tissue damages with adult stem cell mediated tissue engineering therapy in both animal models and patients has demonstrated the feasibility of using this technique for tissue/organ regeneration. Further studies in allogeneic adult stem cells and the establishment of universal embryonic stem cells will be critical for the industrialization of tissue engineering in future.

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells.

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 106 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6~8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6~8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.