Estrogen promotes Epithelial ovarian cancer cells proliferation via down-regulating expression and activating phosphorylation of PTEN.

Epithelial ovarian cancer (EOC) is the most common of cancer death among malignant tumors in women, its occurrence and development are strongly linked to estrogen. Having identified the phosphatase and tensin homologue (PTEN) is a potent tumor suppressor regulating cell proliferation, migration, and survival. Meanwhile, there is a correlation between PTEN protein expression and estrogen receptor expression in EOC. However, no study has amplified on the molecular regulatory mechanism and function between estrogen and PTEN in the development of EOC. In this research, we found that PTEN shows a low expression level in EOC tissues and estrogen decreased PTEN expression via the estrogen receptor 1 (ESR1) in EOC cells. Knockdown of PTEN enhanced the proliferation and migration level of EOC cells driven by estrogen. Moreover, PTEN was also phosphorylated by G protein-coupled receptor 30 (GPR30)-Protein kinase C (PKC) signaling pathway upon estrogen stimulation. Inhibiting the phosphorylation of PTEN weakened the proliferation and migration of estrogen induced-EOC cells estrogen and decreased the phosphorylation of Protein kinase B (AKT) and Mammalian target of rapamycin (mTOR). These results indicated that estrogen decreased PTEN expression level via the ESR1 genomic pathway and phosphorylated PTEN via the GPR30-PKC non-genomic pathway to activate the PI3K/AKT/mTOR signaling pathway, thereby determining the fate of EOC cells.

Adult-type granulosa cell tumor of the ovary.

Adult-type Granulosa Cell Tumor of the Ovary (AGCT) is a relatively rare subtype of ovarian cancer, accounting for 2-4% of all ovarian cancer. AGCT originates from proliferating normal preovulatory granulosa cells (GCs) and retains several features of those GCs. The hormonal features of AGCT explain the clinical manifestations and provide reliable markers for early diagnosis and recurrence prediction of the disease. Most AGCT patients are diagnosed at an early stage and usually demonstrate a better prognosis than patients with other types of ovarian cancer. Surgery is crucial for both initial and post-relapse treatments, whereas adjuvant therapy is still in the exploratory stage. In 2009, a population-based screening makes an exciting step, about 97% of AGCT has somatic missense mutations in the transcription factor FOXL2 gene and the FOXL2 mutation is considered to be a molecular characteristic of AGCT. Unfortunately, the FOXL2 mutation does not fully explain the development of AGCT. Ongoing research is focusing on signalling pathways in the molecular pathogenesis of AGCT to identify the possible pathogenetic factors and signal transduction pathways and provide a theoretical basis for targeted treatment. Postoperative recurrence of ovarian AGCT is common and is associated with a high mortality rate, which necessitates regular follow-up. The life management of postoperative patients is also crucial, which requires multidisciplinary experts to design recurrence treatment from the perspective of patients and implement meaningful treatment measures.

The Modified Rectangle Flap Epicanthoplasty: A Novel and Individualized Design.

BACKGROUND: The epicanthal fold is ordinary in the eyelids of Asians, and the aesthetic appearance of eyelid surgery could be reduced and undermined; thus, medial epicanthoplasty is commonly performed to eliminate the effect of the epicanthal fold with less scarring. At present, there are a lot of techniques that have been described for the treatment of epicanthal fold. The potential problems, however, such as visible scar or under correction in the medial canthus area are challenges to surgeons. The purpose of our study was to explore a novel and individualized design using a modified rectangle flap with acceptable functional and aesthetic outcomes. METHODS: From January 2017 to January 2018, epicanthoplasty was performed for 40 patients by using a modified rectangle flap. All patients underwent double-eyelid surgery at the same time when they needed it. The evaluation criteria included the intercanthal distance (ICD), interpupillary distance (IPD), the ratio of ICD to IPD (ICD ratio), scar visibility, and cosmetic results. RESULTS: From January 2017 to January 2018, the modified rectangle flap method was carried out on 40 patients, who were evaluated at follow-up from 7 to 15 months. The average intercanthal length was 36.9 ± 2.2 mm preoperatively and decreased significantly to 31.5 ± 1.8 mm postoperatively, 7 months after the surgery (P < 0.01). The excellent cosmetic results, in terms of an open medial canthus, were observed during follow-up periods, with no definite recurrence, hypertrophic scar, or injury of the lacrimal apparatus. The inner canthus and lacrimal caruncle are fully exposed with an invisible scar. Both the patients and the surgeon judged that the aesthetic outcomes were excellent or good. CONCLUSIONS: This modified rectangular flap is an effective and personalized method of correcting the medial folds that leave no additional scar in the medial canthal area, and the procedure meets the patient's aesthetic expectations. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

17ß-Estradiol via Orai1 activates calcium mobilization to induce cell proliferation in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal estrogen-sensitive gynecological cancer. Studies have reported that estrogen induces rapid cellular calcium mobilization in cells and can determine the fate of a cell. We found that estrogen increased the calcium release-activated calcium channel modulator 1 (Orai1) protein expression levels in SK-OV-3 cells. However, to date, there has been no research on the functional relationship and molecular mechanism of estrogen-regulating Orai1 during EOC development. In our study, Orai1 had a high expression level in high-grade serous ovarian tumor tissues and SK-OV-3 cells. Estrogen promoted cell proliferation and migration while inhibiting cell apoptosis in SK-OV-3 cells. Orai1 silencing suppressed estrogen-induced cell migration and proliferation. Overexpression of Orai1, however, enhanced the ability of 17ß-estradiol (E2) to exert its function. Estrogen induced rapid calcium influx in SK-OV-3 cells. Knockdown of Orai1 in SK-OV-3 cells blocked E2-induced stored-operated Ca2+ influx. The messenger RNA expression of caspase 3, matrix metallopeptidase 1, and cyclin-dependent kinase 6 were regulated via Orai1 under E2 treatment. Our results suggest that estrogen, by regulating Orai1, induced calcium influx to determine cell fate.

A moldable thermosensitive hydroxypropyl chitin hydrogel for 3D cartilage regeneration in vitro and in vivo.

Because of poor self-repair capacity, the repair of cartilage defect is always a great challenge in clinical treatment. In vitro cartilage regeneration provides a potential strategy for functional reconstruction of cartilage defect. Hydrogel has been known as an ideal cartilage regeneration scaffold. However, to date, in vitro cartilage regeneration based on hydrogel has not achieved satisfactory results. The current study explored the feasibility of in vitro 3D cartilage regeneration based on a moldable thermosensitive hydroxypropyl chitin (HPCH) hydrogel and its in vivo fate. The thermosensitive HPCH hydrogel was prepared and characterized. Goat auricular chondrocytes were encapsulated into the HPCH hydrogel to form a chondrocyte-hydrogel construct. The constructs were injected subcutaneously into nude mice or molded into different shapes for in vitro chondrogenic culture followed by in vivo implantation. The results demonstrated that the HPCH hydrogel possessed satisfactory gelation properties (gelation time < 18 s at 37 °C), biocompatibility (cell amount almost doubled within one week), and the ability to be applied as an injectable hydrogel for cartilage regeneration. All the constructs of in vitro culture basically maintained their original shapes (in vitro to initial: 110.8%) and displayed typical cartilaginous features with abundant lacunae and cartilage specific matrix deposition. These in vitro samples became more mature with prolonged in vivo implantation and largely maintained the original shape (in vivo to in vitro: 103.5%). These results suggested that the moldable thermosensitive HPCH hydrogel can serve as a promising scaffold for cartilage regeneration with defined shapes in vitro and in vivo. STATEMENT OF SIGNIFICANCE: Because of avascular and non-nervous characteristic of cartilage, in vitro regeneration plays an important role in reconstructing cartilage function. Hydrogel has been known as an ideal cartilage regeneration scaffold. However, to date, in vitro cartilage regeneration based on hydrogel has not achieved satisfactory results. The current study demonstrated that the chondrocyte-hydrogel construct generated by high density of chondrocytes encapsulated into a thermosensitive HPCH hydrogel could successfully regenerate in vitro typical cartilage-like tissue with defined shapes and further mature to form homogeneous cartilage with their original shapes after in vivo implantation. The current study indicated that the moldable thermosensitive HPCH hydrogel could serve as a promising scaffold for in vitro and in vivo cartilage regeneration with different shapes.

Estrogen regulates forkhead transcription factor 2 to promote apoptosis of human ovarian granulosa-like tumor cells.

Granulosa cell tumors of the ovary (GCTs) are the predominant form of ovarian stromal tumors and can lead to abnormally secreted estrogen hormones. Studies have reported that forkhead transcription factor 2 (FOXL2) inhibits estrogen synthesis and its gene mutation can lead to GCTs. We unexpected found that estrogen also regulates the expression level of FOXL2. High-dose estrogen increased the expression of FOXL2 in ovarian-like granulosa (KGN) cells at both the mRNA and protein levels. However, no research has reported on the molecular regulatory mechanism and function between estrogen and FOXL2 in the development of GCTs. In this research, FOXL2 was highly expressed in KGN cells and ovarian stromal tumor tissues. Deletion of FOXL2 increased the estrogen secretion in KGN cells. In turn, high-dose estrogen increased the FOXL2 expression levels. FOXL2 was phosphorylated by GPR30 (G protein coupled receptor)-Protein kinase C (PKC) signaling pathway upon estrogen stimulation. Estrogen inhibited cell migration and proliferation, while promoting cell apoptosis. Deletion of FOXL2 inhibited the influence of estrogen on cell proliferation, migration, and apoptosis. Results suggest that estrogen via regulating FOXL2 suppresses cell proliferation and induces cell apoptosis.

BMP2/7 heterodimer enhances osteogenic differentiation of rat BMSCs via ERK signaling compared with respective homodimers.

Bone morphogenetic protein (BMP)2/7 heterodimer shows greater efficacy in enhancing bone regeneration. However, the precise mechanism and the role of mitogen-activated protein kinase (MAPK) signaling network in BMP2/7-driven osteogenesis remain ambiguous. In this study, we evaluated the effects of BMP2/7 heterodimers on osteoblastic differentiation in rat bone marrow mesenchymal stem cells (BMSCs), with the aim to elaborate how MAPKs might be involved in this cellular process by treatment of rat BMSCs with BMP2/-7 with a special signal-pathway inhibitor. We found that BMP2/7 heterodimer induced a much stronger osteogenic response in rat BMSCs compared with either homodimer. Most interestingly, extracellular signal-regulated kinase (ERK) demonstrated a highly sustained phosphorylation and activation in the BMP2/7 heterodimer treatment groups, and inhibition of ERK cascades using U0126 special inhibitor that significantly reduced the activity of ALP and calcium mineralization to a substantial degree in rat BMSCs treated with BMP2/7 heterodimers. Collectively, we demonstrate that BMP2/7 heterodimer shows a potent ability to stimulate osteogenesis in rat BMSCs. The activated ERK signaling pathway involved in this process may contribute partially to an increased osteogenic potency of heterodimeric BMP2/7 growth factors.

Transfection of adenovirus-mediated mircoRNA-126 gene into infant hemangioma endothelial cells in vitro.

Objective: To investigate the effects of microRNA-126 (miR-126) overexpression on hemangioma endothelial cells (HemECs). Methods: An adenoviral vector containing the miR-126 gene was constructed. HemECs were passaged and expanded and adenovirus-mediated green fluorescent protein (GFP) gene was transfected in vitro. The infection efficiency of adenovirus vector to HemECs was tested by Ad-GFP infection procedure. GFP expression efficiency was observed using a fluorescence microscope and flow cytometry was used to determine the best virus multiplicity of infection (MOI). The experiment was divided into the blank group, AD-GFP group, and AD-miR-126 group. The miR-126 group was transfected into HemECs in vitro with adenovirus-mediated miR-126 gene under optimal MOI conditions. RT-PCR was applied to detect expression of miR-126 gene in cells. The influence of recombinant adenovirus on cell activity was evaluated by CCK-8 assay. Flow cytometry was utilized to detect cell cycle and apoptosis. Results: HemECs could be effectively infected by adenovirus containing GFP gene in vitro, the transfection efficiency had the dose-effect relationship with multiplicities of infection (MOI). When MOI was 400, the infection efficiency was more than 90%. miR-126 expression in HemECs was significantly enhanced in miR-126 group (P<0.05). Compared to the control group, cell proliferation was significantly enhanced (P<0.05) and induced S-phase arrest significantly (P<0.05) when miR-126 was upregulated. In addition, compared with the control group, the early apoptotic rate was significantly decreased by upregulating miR-126 (P<0.05). Conclusion: miR-126 overexpression can successfully promote proliferation and inhibit apoptosis of HemECs. This work will provide the theoretical and experimental basis for further transplantation study in vivo.

Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering.

Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

[Experimental study of chondrogenesis in vitro by co-culture of bone marrow stromal cells and chondrocytes].

OBJECTIVE: To investigate the feasibility of chondrogenesis in vitro with bone marrow stromal cells (BMSCs) induced by the co-cultured chondrocytes. METHODS: The BMSCs and chondrocytes were separated from pig and cultured. The supernatant of chondrocytes was used as the inducing solution for BMSCs from the 2nd generation. 7 days later, samples were taken and underwent immunohistochemistry and RT-PCR for detection of the expression of specific type II cartilage collagen, type II collagen and aggrecan mRNA. The cultured BMSCs and chondrocytes were mixed at a ratio of 8:2 (BMSC: cartilage cell) and were inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold at the final concentration of 5.0 x 10(7)/ml. The cartilage cells and BMSCs were also inoculated separately at the same concentration as the positive and negative control. Pure cartilage cells at 20% of the above mentioned concentration (1.0 x 10(7)/ml) were used as the low concentration cartilage cell control group. Samples were collected 8 weeks later. General observations, wet weight, glycosaminoglycans (GAGs) determination and histological and immunohistochemistry examinations were performed. RESULTS: The expression of type II collagen, type II collagen and aggrecan mRNA were positive in induced BMSCs. In the co-cultured group and the positive control group, pure mature cartilage was formed after 8 weeks of culture in vitro, and the size and shape of the scaffold were maintained. The newly formed cartilage in the two groups were almost the same in appearance and histological properties. The immunohistochemistry results indicated that the cartilage cells of the two groups all expressed ample cartilage-specific type II collagen. The average wet weight and GAG content in the co-cultured group reached more than 70% of those in positive control group. Only an extremely small amount of immature cartilage tissues was formed in local regions in pure BMSC group, and the scaffold was obviously shrunk and deformed. Although the wet weight of newly generated cartilage tissue in the low concentration cartilage cell group reached 30% of that in positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly formed cartilage was obviously less than that in the co-culture group and the positive control group. CONCLUSIONS: Chondrocytes can provide a micro-environment for the formation of cartilage, and also effectively induce BMSC to differentiate into chondrocytes and form tissue-engineered cartilage in vitro.

[Complex of auto-cartilage grafting and silicone implant for open nasal plasty].

OBJECTIVE: To investigate the surgical methods and outcome of reshaping the nose by using autologous cartilage grafting-silicone gel complex combined with trimming the lower lateral cartilages and thinning the superfluous tissue of the tip. METHODS: Between May 2006 and July 2008, 36 patients with ugly nose shape received open nasal plasty by thinning the superfluous tissue and trimming the lower lateral cartilages combined with implant of auto-cartilage and silicone gel complex. There were 3 males and 33 females with an average age of 23 years (range, 18-36 years), including 20 cases of hypertrophy and obtuse round of nasal tip, 10 cases of flat of nasal tip, 2 cases of slight nostril exposure, and 4 cases of small whole nose with hypertrophy of nasal tip. Among them, 8 cases received 2-time operations. RESULTS: All incisions achieved healing by first intention. No deformation and complication occurred at donor sites of cartilage. The appearance, contour, color, and touch sensation of the nose were satisfactory and no complications of prosthesis exposure and skin redness of the nasal tip occurred. At 3-5 months after operation, the appearance of the nasal tip was satisfactory when part of the soft tissue was absorbed. Thirty-two patients were followed up 3-12 months (6 months on average), who were satisfied with the appearance of nose with good correct rate. CONCLUSION: Nasal plasty by using auto-cartilage grafting and silicone implant combined with trimming the lower lateral cartilages and thinning the superfluous tissue of the tip is an effective method especially for round or bulbous nasal tip.

Effects of chondrogenic microenvironment on construction of cartilage tissues using marrow stromal cells in vitro.

OBJECTIVE: To investigate whether it is feasible to use the chondrogenic microenvironment provided by cartilage cells to construct cartilage tissues in vitro with bone marrow stromal cells (BMSC). MATERIALS AND METHODS: We isolated and cultured BMSC and cartilage cells from Sprague Dawley rats (SD rats). The supernatant of cartilage culture was used as inducing solution to cause differentiation of BMSC from the second generation of cells cultured in vitro. Cells were examined seven days later, using immunohistochemistry to determine the expression of collagen specific to type II cartilage. RT-PCR was used to detect the expression of type II collagen and aggrecan mRNA. BMSC and cartilage cells were isolated from SD rats and cultured in vitro. The BMSC and cartilage cells in culture were mixed evenly in an 8:2 ratio and inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold to a final concentration of 5.0x10(7) cells/ml. PGA/PLA preparations with pure cartilage cells or pure BMSC served as the positive and negative controls, respectively. The control group of low-concentration cartilage cells consisted of PGA/PLA preparations containing cartilage cells at 20% of the above mentioned concentration (1.0x10(7) cells/ml). Samples were collected eight weeks later, at which time general observations, wet weight, and glycosaminoglycan (GAG) levels were determined, and histological and immunohistochemical examinations were performed. RESULTS: Immunohistochemistry showed the induction of BMSC type II collagen, and RT-PCR indicated the expression of type II collagen and aggrecan mRNA. In the mixed-cell group and the positive control group, pure mature cartilage cells were produced after eight weeks of culture in vitro, and the size and shape of the scaffold were maintained throughout the culture period. The two groups gave rise to newly generated cartilage cells essentially identical in appearance and histological properties. The immunohistochemical results showed that the cartilage cells of both groups expressed abundant cartilage-specific type II collagen. The average wet weight and GAG content were more than 70% of the values in the positive control group. Only an extremely small amount of immature cartilage tissue formed in local regions in the BMSC-only sample, and the scaffold was obviously shrunken and deformed. Although the wet weight of newly generated cartilage tissue in the low-concentration cartilage cell sample reached 30% of the value of the positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly generated cartilage was obviously less than in the co-culture and positive control groups. CONCLUSIONS: Cartilage cells can provide a microenvironment for cartilage formation to some extent, and also effectively induce BMSC to differentiate into cartilage cells and form tissue-engineered cartilage in vitro.

[Study of chondrocytic differentiation of bone marrow stem cells with the supernatant of chondrocytes in vitro].

AIM: To induce bone marrow stem cells(BMSC) of rats to differentiate directionally towards chondrocytes in vitro and identify the differentiated cells. METHODS: BMSC and chondrocytes were isolated from SD rats and cultured in vitro. The supernatant of chondrocytes was collected and used to induce transformation of BMSC from the second passage. After 7 days of induction, specific markers of differentiation of chondrocytes were detected by the appearance of toluidine blue staining, Masson staining, immunohistochemistry detection of collagen type II, and RT-PCR detection of collagen type II and aggrecan mRNA. RESULTS: After 7 days, the induced BMSC changed into triangle or polygonal shape from spindle shape. Specific markers of chondrocytes were positive in the appearance of toluidine blue staining, Masson staining, immunohistochemistry detection of collagen type II, and RT-PCR detection of collagen type II and aggrecan mRNA. CONCLUSION: The supernatant of chondrocytes can induce BMSC to differentiate into chondrocytes in vitro.

[Experimental study of in vitro chondrogenesis by co-culture of bone marrow stromal cells and chondrocytes].

OBJECTIVE: Chondrogenic microenvironments play a very important role in chondrogenesis of bone marrow stromal cells (BMSC). This study explored the feasibility of in vitro chondrogenesis by co-culture of BMSC and chondrocytes so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of BMSC and thus promote in vitro chondrogenesis of BMSC. METHODS: Porcine BMSC and auricular chondrocytes were in vitro expanded respectively and then were mixed at a ratio of 8:2 (BMSC:chondrocyte). 200 microl mixed cells(5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 9 mm in diameter and 3 mm in thickness, as co-culture group. Chondrocytes and BMSC with the same cell number were seeded respectively onto the scaffolds as positive control (chondrocyte group) and negative control (BMSC group). 200 microl chondrocytes (1.0 x 10(7)/ml, equal to the chondrocyte number of co-culture group) alone were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, average wet weight measurement, glycosaminoglycan (GAG) quantification, histology and immunohistochemistry were used to evaluate the results. RESULTS: Cells in all groups had fine adhesion to the scaffolds and could secrete extracellular matrix. In both co-culture group and positive control group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture and formed homogenous mature cartilage after 8 weeks of in vitro culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features, and abundant type II collagen was also detected by immunohistochemistry in both groups. The average wet weight and GAG content of co-culture group were both more than 80% of those of positive control group. In negative control group, however, the constructs shrunk gradually during in vitro culture and cartilage-like tissue could only be observed at the edge area of the construct. In low concentration chondrocyte group, the constructs also shrunk gradually during in vitro culture and the average wet weight was below 40% of that of the positive control group although histology showed a small amount cartilage formation. CONCLUSION: Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of BMSC and thus promote the in vitro chondrogenesis of BMSC.

[Repairing porcine knee joint osteochondral defects at non-weight bearing area by autologous BMSC].

OBJECTIVE: To test the possibility of using bone marrow stromal cells (BMSC) and biodegradable polymers to repair articular osteochondral defects at non-weight bearing area of porcine knee joints. METHODS: Bone marrows were harvested from 18 hybrid pigs. BMSC were cultured and in vitro expanded and induced with dexamethasone (group A) or with dexamethasone and transforming growth factor-beta1 (TGF-beta1) (group B) respectively. Immunohistochemistry and RT-PCR were used to evaluate chondrogenic differentiation of induced cells. Part of BMSC of 2 animals were retrovirally-labeled with green fluorescent protein (GFP). After induction and label, cells were seeded on a construct of polyglycolic acid (PGA) and polylactic acid (PLA) and co-cultured for 1 week before implantation. Total 4 osteochondral defects (8 mm in diameter, 5 mm in depth) in each animal were created at the non-weight bearing areas of knee joints on both sides. The defects were repaired with dexamethasone induced BMSC-PGA/PLA construct in group A, with dexamethasone and TGF-beta1 induced BMSC-PGA/PLA construct in group B, with PGA/PLA construct alone (group C) or left untreated (group D) as controls. Animals were sacrificed at 3 months (n = 6) or 6 months (n = 10) post-repair. Gross observation, histology, glycosaminoglycan (GAG) quantification and biomechanical test were applied to analyze the results. The two animals with GFP-labeled cells were sacrificed at 7 months post-repair to observe with confocal microscope the distribution of GFP-labeled cells in repaired tissue. RESULTS: Stronger expression of type II collagen and aggrecan were observed in BMSCs induced with both dexamethasone and TGF-beta1. At both time points, Gross observation and histology showed that the defects in most of group A were repaired by engineered fibrocartilage and cancellous bone with an irregular surface, minority defects were repaired by engineered hyaline cartilage and cancellous bone. However, in most of group B, the defects were completely repaired by engineered hyaline cartilage and cancellous bone. No repair or only fibrous tissue were observed in groups C and D. Besides, the compressive moduli of repaired cartilage in groups A and B reached 30.37% and 43.82% of normal amount at 3 months and 62.69% and 80.27% at 6 months respectively, which was further supported by the high levels of GAG contents in engineered cartilage of group A (78.03% of normal contents) and group B (no statistical difference from normal contents). More importantly, confocal microscope revealed the presence of GFP-labeled cells in engineered cartilage lacuna and repaired underlying cancellous bone. CONCLUSION: The results demonstrated that implanted BMSC can differentiate into either chondrocytes or osteoblasts at different local environments and repair a complex articular defect with both engineered cartilage and bone. TGF-beta1 and dexamethasone in vitro induction can promote chondrogenic differentiation of BMSC and thus improve the results of repairing articular defects.

[Influence of mechanical stress on chondrogenesis of in vitro cultured porcine bone marrow stem cells: a preliminary study].

OBJECTIVE: To evaluate the influence of mechanical stress on chondrogenesis of in vitro cultured porcine bone marrow stem cells (BMSC). METHODS: Porcine BMSC of passage 2 were seeded onto a cylinder-shaped PGA/PLA scaffold, 8mm in diameter and 3mm in thickness, at a density of 5 x 10(7)/cm(3). After the cell-scaffold constructs were cultured for one week, the primary medium, high-glucose DMEM medium with 10% fetal bovine serum (FBS), was replaced by chondrogenically inductive medium containing TGFbeta(1) (10 ng/ml), IGF-I (50 ng/ml), and dexamethasone (40 ng/ml) in addition to DMEM+10% FBS. The constructs were randomly divided into three groups according to the imposed stress: experimental group A in which a centrifugal stress was imposed at 100 g, 30 min, 2/d; experimental group B in which a rotative stress was imposed at 80 rpm, 8 h/d by a shaker; and control group in which the constructs were statically cultured. The gross view, histology, histochemistry, immunohistochemistry and glycosaminoglycan (GAG) content were evaluated after 4 and 8 weeks respectively. RESULTS: Four weeks later, the constructs in both experimental groups maintained their original sizes and shapes. Histology showed nodular lacuna-like structures, in company with GAG deposition and collagen synthesis. In addition, collagen type II was detected by immunohistochemistry. In the control group, however, the constructs shrunk to a little smaller size than those in the experimental groups, and histological staining showed a little amount of lacuna. Eight weeks later, the constructs in both experimental groups still maintained the original sizes and shapes with good elasticity. HE staining showed massive lacuna-like structures in most areas of the construct and extracellular matrix deposited evenly. Fibrous tissues were only observed in some areas. Safranin-O staining showed massive GAG formation and Masson staining showed much more collagen formation than those in the control group. Immunohistochemical staining of collagen type II showed strong positive expression. In the control group the constructs showed massive fibrous tissues, with a small amount of lacuna-like structures in the peripheral areas. GAG contents in the 2 experimental groups were 5.98 mg/g and 5.62 mg/g respectively, both significantly higher than that in the control group (4.73 mg/g) without a difference between the 2 experimental groups. CONCLUSION: Mechanical stress promotes chondrogenesis and cartilage maturation of BMSC in vitro.