Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta1 (TGF-beta1) alter connexin 43 phosphorylation in MC3T3-E1 Cells.

BACKGROUND: Bone morphogenetic proteins (BMPs) and transforming growth factor-betas (TGF-betas) are important regulators of bone repair and regeneration. BMP-2 and TGF-beta1 have been shown to inhibit gap junctional intercellular communication (GJIC) in MC3T3-E1 cells. Connexin 43 (Cx43) has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-beta1 to investigate a possible mechanism for the inhibition of GJIC. RESULTS: Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-beta1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. CONCLUSIONS: BMP-2 and TGF-beta1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-beta1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells.

Effect of serial passage on gene expression in MC3T3-E1 preosteoblastic cells: a microarray study.

The osteoblastic function of mouse preosteoblastic MC3T3-E1 cells, as measured by alkaline phosphatase activity and osteocalcin secretion, decreases after serial passage. To uncover genes responsible for decreased osteoblastic function in high-passage cells, we have studied passage-dependent change of gene expression in MC3T3-E1 cells. Changes in the expression pattern of 2000 selected genes were examined simultaneously by comparing mRNA levels between MC3T3-E1 cells at passage 20 and passage 60 using the cDNA microarray analysis. Significant changes in the steady-state abundance of 27 mRNAs were observed in response to different passage numbers, including 17 known genes, 4 ESTs with homology to known genes, and 6 genes with no previously described function or homology. Northern blot analysis was used to verify and quantify the expression of selected genes, and revealed a significant higher level of up- and down-regulation compared to microarray data. These results indicate the existence of a significant change in gene expression in osteoblastic cells undergoing serial passages. Such changes might be responsible for a reduction in bone regeneration in older osteoblasts. Potential roles of selected genes in bone aging are discussed.

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.

Staged skin and subcutaneous excision for lymphedema: a favorable report of long-term results.

Numerous surgical procedures have been proposed for the management of lymphedema. The postoperative results vary, and unfortunately none of the procedures are curative. As a result, some degree of recurrence of leg edema is seen in all patients postoperatively. Reported here is a long-term follow-up of patients with lower extremity lymphedema managed by skin and subcutaneous tissue excision. Thirty-eight patients (6 male; 32 female) with lower extremity lymphedema have been followed up for an average of 14 (3 to 27) years after staged subcutaneous excisions performed beneath skin flaps. Seven patients had been treated previously by other procedures. Of the 38 lymphedema patients, 10 patients developed edema after pelvic or groin ablative surgery, radiation therapy, or both. Results were documented by various methods: physical examination, circumferential measurements, volume displacement, serial photography, lymphoscintigraphy, and patient survey. Of these, it is believed that photographs are the easiest and as representative as any other method, all of which have great variability. Of the 38 patients, 30 patients had significant and long-lasting reduction in extremity size associated with improved function and extremity contour. Episodes of recurrent cellulitis were reduced or completely eliminated. No differences in the long-term results were seen in patients with acquired as opposed to congenital lymphedema. Men did not have as much improvement as women. Two patients had no change in leg swelling, and six patients (three men) had progressive swelling after surgery. Partial wound separation occurred immediately postoperatively in one patient, and three patients had loss (less than 2 cm) of the skin flap, all in the ankle region. None of these instances required further surgery, and no other significant complications were encountered. Staged skin and subcutaneous excision beneath skin flaps appears to provide long-lasting improvement for lower extremity lymphedema, regardless of cause, in the majority of patients treated.

Laparoscopic aortobifemoral bypass.

PURPOSE: To determine the potential benefits of applying laparoscopic techniques for the intraabdominal insertion of aortofemoral grafts and to compare results with those of conventional surgery. METHODS: Having previously demonstrated the feasibility of a totally laparoscopic aortofemoral bypass technique using carbon dioxide peritoneal insufflation in a porcine model, we now report our first human experience with this laparoscopic technique in a 49-year-old man. RESULTS: The patient's postoperative course was marked by his minimal requirements for analgesia, early ambulation, and discharge from the hospital in the morning of the third postoperative day. CONCLUSIONS: The benefits of a laparoscopic approach to aortobifemoral bypass grafting in terms of financial savings and earlier rehabilitation in this patient was significant. This less-invasive procedure warrants further investigation.

Laparoscopic aortobifemoral bypass: a case report.

In the past six years, laparoscopic surgery has gained widespread acceptance by both surgeon and patient. When compared to open surgical approaches, laparoscopic techniques for abdominal procedures lessen postoperative pain and morbidity, improve cosmesis, reduce hospital stay, facilitate early rehabilitation and return to normal activities. The application of laparoscopic techniques to intra-abdominal vascular procedures can be expected to provide similar benefits over conventional surgery.

Evaluation of a novel osteogenic factor, bone cell stimulating substance, in a rabbit cranial defect model.

A novel osteogenic factor, bone cell stimulating substance (BCSS), was recently isolated from bovine bone. Unlike the bone morphogenetic proteins, which are generally 20- to 40-kDa glycoproteins belonging to the transforming growth factor-beta superfamily, BCSS is a 2.5-kDa, water-soluble polypeptide. This study evaluated the osteogenic potential of BCSS in combination with coralline-derived porous hydroxyapatite to reconstruct a large, nonhealing cranial defect in the rabbit. Twenty-four rabbits underwent a 16 x 20 mm full-thickness (extradural) excision of the parietal bones and were divided into six groups of four rabbits each. Group 1 through 5 were reconstructed with 16 x 20 x 1.5 mm hydroxyapatite implants treated with: (1) 20 osteogenic units (424 micrograms) BCSS, (2) 8 osteogenic units (170 micrograms) BCSS, (3) 4 osteogenic units (85 micrograms) BCSS, (4) 0 osteogenic units (424 micrograms) inactive BCSS analog, or (5) left untreated. Group 6 was left unreconstructed. Implants were harvested at 12 weeks and analyzed for percentage of lamellar bone formation by a computerized microscope video image analysis system. Groups reconstructed with BCSS-treated hydroxyapatite implants demonstrated more bone ingrowth than did the control hydroxyapatite groups not treated with BCSS (inactive BCSS analog or untreated). Linear regression dose-response analysis indicated an average 3.2 percent increase in bone ingrowth for a 10-U increase in BCSS (p = 0.043). The unreconstructed control group demonstrated no healing. With a molecular mass of only 2.5 kDa and its identity not completely known, BCSS is a novel factor that seems to possess osteogenic potential similar to that of previously investigated osteogenic proteins.

Transforming growth factor-beta, osteogenin, and bone morphogenetic protein-2 inhibit intercellular communication and alter cell proliferation in MC3T3-E1 cells.

Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues-processes which are regulated, in part, by peptide growth factors, including transforming growth factor-beta (TGF-beta) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-beta and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3-E1 cells were cultured in the presence of bone morphogenetic protein-2 (BMP-2) or TGF-beta for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by 3H-thymidine (3H-TdR) incorporation. After times ranging from 6 to 48 hr, BMP-2 significantly inhibited uptake of 3H-TdR at doses of 50-800 ng/ml. Similarly, TGF-beta inhibited uptake of 3H-TdR at doses of 2-32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell-cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 microgram/ml) or higher dose of TGF-beta (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP-2 and TGF-beta on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-beta (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP-2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose-dependent effects of BMP-2 and TGF-beta on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-beta at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC50) calculated for BMP-2 and TGF-beta at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF-beta. The observation may suggest that TGF-beta may have effects upon cytoskeletal elements in osseous tissues.

Growth factors in surgery.

Surgical reconstruction of functional and aesthetic defects is often compromised by donor-tissue limitations and wound-healing constraints--problems that can potentially be overcome by peptide growth factor therapy. The effectiveness of growth factor therapy in animal models and in limited human clinical trials has been realized. Epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), platelet-derived wound-healing formula (PDWHF), transforming growth factor (TGF), the bone morphogenetic proteins (BMPs), and general considerations of growth factor therapy are reviewed.

Surgical techniques in breast conservation.

The surgical treatment of breast cancer has changed dramatically in the last 30 years. The era of the Halsted radical mastectomy has passed, and less deforming surgeries have come into use. Partial mastectomy in association with axillary lymph node dissection has become a viable alternative for stage 1 and 2 carcinomas; more advanced tumors may be treated with breast conservative surgery when neoadjuvant chemotherapy is utilized. Further, the use of mammography in screening for breast cancer has led to an increase in the diagnosis of ductal carcinoma in situ (DCIS), another lesion for which breast conservation is often indicated.

Lipedema: a clinical entity distinct from lymphedema.

In a review of 250 cases of lymphedema of the lower extremity, 9 patients were noted to share unique similarities in their history and physical findings. Although these patients had mild swelling in their pretibial areas and were all referred with a diagnosis of lymphedema of the legs, their findings differed significantly from the usual patient with either congenital or acquired lymphedema. Notably, the lower extremity swelling was always bilateral and symmetrical in nature and never involved the feet. Skin changes characteristic of lymphedema were not found, and consistent fat pads were present anterior to the lateral malleoli in each patient. These findings are representative of a clinical entity known as lipedema, which is distinct from lymphedema and for which treatment may be different.