[Creutzfeldt-Jakob Disease and Lyodura:A Special Reference to Prion Disease Control in the Field of Neurosurgery].

In Japan, 156 cases of dura mater-transplanted Creutzfeldt-Jakob disease(dCJD)with a history of Lyodura transplantation have been confirmed until February 2022, with only a few new cases still being identified. The history of Lyodura transplantation is one involving a neurosurgical procedure. The cumulative global number of cases of bovine spongiform encephalopathy-related variant CJD(BSE-related vCJD), which has shaken societies around the world, is 232 as of 2019. Thus, the impact of dCJD on the society in Japan needs no explanation. Thanks to the world's concerted efforts in research and countermeasures, medically induced prion diseases are finally becoming a thing of the past. However, due to the extremely long incubation period of CJD and the difficulty of tracing the source of infection, immediate action in the event of an outbreak is not possible, and efforts must focus on preventing disease outbreaks. Independent of this, approximately 200 cases of solitary and hereditary prion diseases occur annually in Japan. If neurosurgery must be performed on such patients, secondary transmission of prion disease by neurosurgical instruments must be prevented. Therefore, sterilization methods for neurosurgical instruments are critical, and various measures including sterilization methods have been determined and published by a research group designated by the Japanese Ministry of Health, Labour and Welfare. The sterilization of neurosurgical instruments should comply with the latest guidelines that are published by this study group.

P. gingivalis Lipopolysaccharide Stimulates the Upregulated Expression of the Pancreatic Cancer-Related Genes Regenerating Islet-Derived 3 A/G in Mouse Pancreas.

Although epidemiological studies have shown a relationship between periodontal disease and pancreatic cancer, the molecular mechanisms involved remain unclear. In this study, the effects of systemic administration of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on gene expression were comprehensively explored in mouse pancreas that did not demonstrate any signs of inflammation. PG-LPS was prepared in physiological saline and intraperitoneally administered to male mice at a concentration of 5 mg/kg every 3 days for 1 month. After extracting total RNA from the excised mice pancreas, a comprehensive DNA microarray analysis of gene expression was performed. Tissue specimens were also subjected to hematoxylin-eosin staining and immunohistochemistry using anti-regenerating islet-derived 3A and G (Reg3A/G) antibody. ImageJ software was used to quantify the area of Reg3A/G positive cells in pancreatic islets by binarizing image date followed by area extraction. The results were compared using Mann-Whitney U test. Data are presented as mean ± standard deviation (SD) with p < 0.05 considered as significant. Reg3G, a gene related to pancreatic cancer, was one of the 10 genes with the highest levels of expression in the pancreas stimulated with PG-LPS. The comprehensive analysis revealed a 73-fold increase in Reg3G expression level in the PG-LPS group when compared with the control group; in addition, the expression level of Reg3A was increased by 11-fold in the PG-LPS group. Image analysis showed that the ratio of Reg3A/G positive cells was higher in the PG-LPS group than the control. Immunostaining showed the presence of Reg3A/G-positive cells in the alpha-cell equivalent areas around the islets of Langerhans in the PG-LPS group. These results support the notion that periodontal disease may be a risk factor for pancreatic cancer.

Gastric cancer deaths by age group in Japan: Outlook on preventive measures for elderly adults.

In February 2013, Japan became the first country in the world to cover Helicobacter pylori eradication for chronic gastritis under its National Health Insurance (NHI) system. Now that eradication therapy is covered by NHI, its usage has increased dramatically, and gastric cancer deaths have begun to decrease. We undertook a detailed epidemiological analysis to investigate effects of expanded NHI coverage for H. pylori eradication therapy on gastric cancer deaths in specific age groups. Numbers of gastric cancer deaths were determined by referencing data from Ministry of Health, Labour and Welfare reports and "Cancer Statistics in Japan - 2018" published by the Foundation for Promotion of Cancer Research. Gastric cancer deaths across all age groups have been clearly decreasing since 2013, but deaths of people aged 80 years and older are still increasing. The number of gastric cancer deaths in people aged in their 80s was 2 times higher than in people aged in their 70s and 4 times higher than in people aged in their 60s. The number of people in their 80s who had an endoscopy was less than half that of people in their 60s and 70s. The eradication therapy has increased dramatically, and gastric cancer deaths are clearly decreasing in Japan. However, this decrease in deaths has not extended to elderly adults aged in their 80s, which suggests that measures to prevent gastric cancer in people aged 80 years and older will be critical to achieving the mission of eliminating gastric cancer in Japan.

Effect on Helicobacter pylori eradication therapy against gastric cancer in Japan.

BACKGROUND: In Japan, there have been approximately 50 000 deaths from gastric cancer annually for over 40 years with little variation. It has been reported that most gastric cancers in Japan are caused by Helicobacter pylori infection. H. pylori eradication therapy was approved for patients with chronic gastritis by the Japanese national health insurance scheme in February 2013 for patients with an endoscopic diagnosis of chronic gastritis is positive for H. pylori. We examined the effect on gastric cancer death rate 4 years after expansion of health insurance coverage. AIM: We conducted an epidemiological study and analyzed trends in prescription for H. pylori eradication therapy. We used the electronic medical claims database from Hokkaido, Japan to evaluate the impact of expansion of national health insurance coverage for H. pylori eradication therapy on deaths from gastric cancer. METHODS: Data on deaths from gastric cancer were obtained from the Japanese Ministry of Health, Labour and Welfare and the Cancer Statistics in Japan (2015). Analysis of electronic claims records was performed using the National Database, mainly focusing on Hokkaido. Prescriptions for H. pylori eradication therapy and the number of patients treated for gastric cancer were also extracted from the Hokkaido database. RESULTS: Approximately 1.5 million prescriptions for H. pylori eradication therapy were written annually. Gastric cancer deaths fell each year: 48 427 in 2013, 47 903 in 2014, 46 659 in 2015, and 45 509 in 2016, showing a significant decrease after expansion of insurance coverage for H. pylori eradication therapy (P<.0001). CONCLUSIONS: Prescriptions for H. pylori eradication therapy increased markedly after approval of the gastritis indication by the national health insurance scheme and was associated with a significant decrease in gastric cancer deaths.

Early experiences with stem cells in treating chronic wounds.

This review provides a thorough and clear discussion on the outcomes of stem cells in treating chronic wounds. With recent technological developments that now allow isolation and culture of stem cells, researchers are able to perform vigorous studies on somatic or adult stem cells. Human and animal stem cell studies are discussed with a focus on the basic process of stem cells in wound healing and the authors' first-hand clinical experience with stem cells used for chronic wound healing.

Noncultured autologous adipose-derived stem cells therapy for chronic radiation injury.

Increasing concern on chronic radiation injuries should be treated properly for life-saving improvement of wound management and quality of life. Recently, regenerative surgical modalities should be attempted with the use of noncultured autologous adipose-derived stem cells (ADSCs) with temporal artificial dermis impregnated and sprayed with local angiogenic factor such as basic fibroblast growth factor, and secondary reconstruction can be a candidate for demarcation and saving the donor morbidity. Autologous adipose-derived stem cells, together with angiogenic and mitogenic factor of basic fibroblast growth factor and an artificial dermis, were applied over the excised irradiated skin defect and tested for Patients who were uneventfully healed with minimal donor-site morbidity, which lasts more than 1.5 years.

Basic fibroblast growth factor is beneficial for postoperative color uniformity in split-thickness skin grafting.

Color changes of visible and exposed body surfaces, such as the face and extremities, after burn injury or surgery, such as skin grafting, flap, or sclerotherapy for vascular malformations, are sometimes a concern. The consequences reduce the satisfaction of both patients and physicians. An easy and reproducible method has not yet been established for an objective analysis of color changes; therefore, we tested a hand-held color analyzer (NF-333; Nippon Denshoku Co. Ltd) with data transport to a computer database and analysis software for posttreatment skin color change. The parameters included L, a, and b, which measure clarity, red, and yellow, respectively. Two groups were prospectively divided with 20 (11 females and nine males) patients per group. One group received skin grafting plus basic fibroblast growth factor (bFGF) spray daily and the other group received only skin grafting. The patients were randomized by the date of their first visit to our hospital. Patients were treated with bFGF on odd days, while patients who came on even days were included in the non-bFGF-treated group. The donor site for skin grafting was the lateral thighs and the thickness was similar in both groups. The results were compared at 1-year posttreatment follow-up. Clinical and objective assessments of the scars were performed 1 to years after complete healing. Color change differentials in comparison with the surrounding skin were lower with bFGF treatment in all parameters (p<0.01), along with clinical assessment with the Vancouver Scar Scale; therefore, the treatment contribute to a better color match with skin grafting postoperatively.

Mesenchymal stem cell therapy for cutaneous radiation syndrome.

Systemic and local radiation injuries caused by nuclear power reactor accidents, therapeutic irradiation, or nuclear terrorism should be prevented or properly treated in order to improve wound management and save lives. Currently, regenerative surgical modalities should be attempted with temporal artificial dermis impregnated and sprayed with a local angiogenic factor such as basic fibroblast growth factor, and secondary reconstruction can be a candidate for demarcation and saving the donor morbidity. Human mesenchymal stem cells and adipose-derived stem cells, together with angiogenic and mitogenic factor of basic fibroblast growth factor and an artificial dermis, were applied over the excised irradiated skin defect and were tested for differentiation and local stimulation effects in the radiation-exposed wounds. The perforator flap and artificial dermal template with growth factor were successful for reconstruction in patients who were suffering from complex underlying disease. Patients were uneventfully treated with minimal morbidities. In the experiments, the hMSCs are strongly proliferative even after 20 Gy irradiation in vitro. In vivo, 4 Gy rat whole body irradiation demonstrated that sustained marrow stromal (mesenchymal stem) cells survived in the bone marrow. Immediate artificial dermis application impregnated with cells and the cytokine over the 20 Gy irradiated skin and soft tissues demonstrated the significantly improved fat angiogenesis, architected dermal reconstitution, and less inflammatory epidermal recovery. Detailed understanding of underlying diseases and rational reconstructive procedures brings about good outcomes for difficult irradiated wound healing. Adipose-derived stem cells are also implicated in the limited local injuries for short cell harvesting and processing time in the same subject.

Human mesenchymal stem cells may be involved in keloid pathogenesis.

BACKGROUND: The pathogenesis of keloid is poorly understood. Although vigorous investigations have attempted to elucidate the mechanisms or causative factors of keloid, there are little data on why keloids are very intractable and recur easily in each patient. METHODS: In an attempt to analyze the possible interaction between human mesenchymal stem cells and keloid-derived fibroblasts, the dual-chamber cell-migration assay, cell proliferation, ultrastructural morphology, and Western blot analysis were used to investigate the production of the extracellular matrices of the coculture. RESULTS: Cell proliferation was not significantly different between keloid-derived fibroblasts and normal dermal fibroblasts during a 4-day observation period. There was a significant cell migration of human mesenchymal stem cells when keloid-derived fibroblasts were placed in the bottom chamber, compared to when normal dermal fibroblasts were placed in the same way in 8-microm diameter pore membranes (190.6 +/- 51.45 and 32.0 +/- 6.20 cells/field, respectively, P < 0.01). With 3-microm diameter pores, the human mesenchymal stem cells migrated in the pores only when the keloid-derived fibroblasts were placed in the bottom chambers (6.4 +/- 3.84 cells/field). Monolayer coculture of human mesenchymal stem cells and keloid-derived fibroblasts demonstrated further functional differentiation, such as collagen secretion and abundant rough endoplasmic reticulum. Western blot analysis of the cells in the modified dual-chamber culture demonstrated most significantly abundant fibronectin expression when the human mesenchymal stem cells contained keloid fibroblasts. CONCLUSION: The results of this study may indicate that human mesenchymal stem cells participate and recruit in keloid pathogenesis by differentiating themselves toward keloid recalcitrant formation and progression.

Combined surgical excision and radiation therapy for keloid treatment.

Various methods have been attempted for the treatment and management of keloids; however, there is little satisfactory clinical evidence in long-term follow ups. Also, there is a preference for occurrence and recurrence in anatomic location. Usually anatomic locations with higher regional tension and more sebaceous glands are inclined toward pathogenesis. Thirty-eight keloids treated with combined surgical excision and postoperative irradiation, using electron beams with only a 10-mm opening by lead shielding, were investigated at a mean follow up of 4.4 +/- 2.5 years (range, 1-9 years) at a single institute. Ten locations such as the ear (n = 6), neck (n = 3), and upper lip (n = 1) were among the craniofacial locations. The hardness of the keloids and posttreatment scars was clinically and objectively tested with the Vancouver scar scale and a durometer, which is often used for the industrial measurement of thread balls and rubber. At a mean of 4.4 +/- 2.5 years of follow up, the clinical characteristics of the scars were significantly better posttreatment as 2.6 +/- 0.5 versus 1.0 +/- 0.6, 3.7 +/- 0.7 versus 1.7 +/- 0.7, 2.9 +/- 0.4 versus 1.3 +/- 0.5, and 2.7 +/- 0.5 versus 1.3 +/- 0.5 (keloid scars versus posttreatment scars: pigmentation, pliability, height and vascularity, respectively, P < 0.01). The durometer readings were significantly lower posttreatment, 15.2 +/- 3.9 versus 7.7 +/- 2.9 (keloid scars versus posttreatment scars, P < 0.01). The recurrence rate was 21.2% overall with none in craniofacial locations. Therefore, the combined treatment of surgical excision and postoperative electron beam irradiation is effective for scar quality and reducing the recurrence rate in long-term follow up.

Acceleration of sensory neural regeneration and wound healing with human mesenchymal stem cells in immunodeficient rats.

The sensory nerve is highly involved in lower extremity wound healing. In diabetic and vascular diseases, impaired nerve function and blood flow delay wound healing. Tissue regeneration using adult stem cells is a targeted therapeutic modality in disorders of nerve and blood supply. Effective delivery using an autologous vascularized fascial flap as a vehicle of stem cells leads to severed sensory nerve recovery, local tissue blood flow, and wound healing. Human MSCs (hMSCs) were transfected with green fluorescent protein (GFP) cDNA and tested for efficiency and proliferation in vitro. The nude rat model with femoral vessel and saphenous nerve severance and ligation was wrapped with a vascularized epigastric flap for GFP-hMSC, fibroblast growth factor-2 (FGF-2), or a combination of both after 2 weeks. Maximum nerve conduction velocity recovered to 70% of the presurgical level in the GFP-hMSC- and FGF-2-treated group at 2 weeks. Blood flow and nerve conduction velocity were positively correlated at 1 week. Wound healing in the ipsilateral paw had significantly improved by 1 week. Histologically, blood vessels and nerves are very organized, and regenerated neuron immunoreactivity of GAP-43 and a nerve regrowth marker of S-100 were remarkable in the human GFP (hGFP)-hMSC and FGF-2-treated group at 2 weeks; therefore, sensory nerve regeneration, blood flow, and wound healing were improved by the administration of stem cells and FGF-2 via a vascularized flap. This may be implicated in clinical denervated and reduced circulation tissue wound healing.

Therapeutic choice for craniofacial venous malformations.

Even though the precise mechanisms related to venous malformation are still unclear, the clinical manifestations sometimes threaten vital signs such as mastication, airway and phonics. Our therapeutic modalities were reviewed, and their effectiveness and related complications were analyzed. Between March, 1998 and February, 2006, 11 patients (15-59 years old; average 32.4 +/- 13.60, 4 women and 7 men) with craniofacial venous malformation were included in this investigation. All cases experienced some kind of surgery at least once during clinical follow-up. Direct puncture scintigraphy with technetium-99m Sn colloid-labeled demonstrated low-flow malformations in all cases. Two cases underwent bone surgery and another two cases had static suspensions for facial nerve paralysis. Blood loss from surgery alone was 1352 +/- 1115.0 mL, simultaneous procedures yielded 400 +/- 244.9 mL blood loss and sclerotherapy alone resulted in 187 +/- 284.8 mL of blood loss (surgery alone versus sclerotherapy alone, P < 0.01). Excellent sclerotherapy cases were when the malformation was localized and the number of sclerotherapies was significantly fewer than good cases (1.3 +/- 0.58 times versus 3.6 +/- 1.15 times, excellent, good, respectively, P < 0.05). Although there are difficulties in understanding the mechanisms and multiple therapeutic interventions are required, there have been satisfactory outcomes so far and the development of better sclerosants or a real-time navigation system may benefit more precise therapeutic effects and lower morbidity.

Attenuation of cysteinyl leukotrienes induces human mesenchymal stem cell differentiation.

Although there are numerous investigations describing bone marrow cells or bone-marrow derived cells at the site of such injuries as bone fractures, infarction and subsequent ischemic reperfusion injury, or cutaneous wounds, little is know about the factors that affect the cells in those clinical situations. Cysteinyl leukotrienes have been extensively investigated in airway diseases that may eventually lead to lung fibrosis; while the engraftment of mesenchymal stem cells have been shown to reverse bleomycin-induced lung fibrosis in vivo. Therefore, we elucidated the involvement of cysteinyl leukotrienes in human mesenchymal stem cell proliferation and differentiation. Human mesenchymal stem cells express the cysteinyl leukotriene type 1 receptor. Various doses of pranlukast, which is a specific cysteinyl leukotriene type 1 receptor antagonist, failed to affect the proliferation of cells; however, 10(-6) M of pranlukast significantly induced cellular cytoplasmic differentiation by showing microvilli sprouting and the emersion of rough endoplasmic reticulum within a 16-hour(s) incubation. Additionally, pranlukast-induced fibronectin protein production by human mesenchymal stem cells. Therefore, attenuation of the cysteinyl leukotriene pathway contributes to human mesenchymal stem cell differentiation and may contribute to modulation of the local injury site.

Elevated circulating leukemia inhibitory factor in patients with extensive burns.

To investigate circulating cytokine responsiveness in major burns in association with the systemic stress response system, we tested hypothalamic-pituitary-adrenal (HPA) axis markers in extensive burn cases treated in the Department of Plastic and Reconstructive Surgery at Nagasaki University. The HPA axis is a major stress response system, and the leukemia inhibitory factor (LIF) may be a potent mediator of the HPA axis; therefore, circulating LIF levels in burn patients were studied. Twenty extensively burned patients (burn surface area, >20%), ie, 10 women and 10 men, 37 to 77 years of age (average: 59.1 +/- 12.10 years), were assessed. Circulating LIF, adrenocorticotropic hormone (ACTH), other inflammatory markers, and 24-hour urinary free cortisol excretion levels were investigated. LIF levels were greater in patients who died than in those who survived (186.1 +/- 80.41, 83.5 +/- 64.49 pg/ml, respectively, P < .001) at 36 hours after injury. ACTH levels were more significantly elevated in fatal cases than in those who survived. (41.3 +/- 8.28, 25.2 +/- 7.84 pg/ml, respectively, P < .0001). Twenty-four hour (24 to 48 hours after injury) pooled urinary free cortisol excretion levels also were significantly greater in fatal cases than in the surviving patient group (235.0 +/- 36.49 microg/day, 69.0 +/- 18.04 microg/day, respectively, P < .0001). The correlation between serum LIF and urine free cortisol was significant (r = .30; P < .01) as was the correlation of serum LIF with plasma ACTH (r = .24; P < .01). Serum LIF as well as HPA axis activity markers is a good marker of disease severity and prognosis in patients with extensive burns.

Early cellular changes of human mesenchymal stem cells and their interaction with other cells.

Cell-to-cell interactions between human mesenchymal stem cells and potential adjacent cells such as endothelial cells, dermal fibroblasts, and epidermal keratinocytes was investigated. A modified dual Boyden chamber assay using 8-microm pores revealed a more powerful chemotactic cell migration of human mesenchymal stem cells toward human epidermal keratinocytes than other cells, such as umbilical artery endothelial cells and dermal fibroblasts, during 16 hours of incubation (336.2+/-52.33, 36.0+/-11.20, and 62.7+/-18.16, cells/field, respectively, p<0.01; comparison between endothelial cells and keratinocytes, and fibroblasts and keratinocytes). Scanning electron microscopy showed human mesenchymal stem cell migration through the pores, with endothelial cells, fibroblasts, or keratinocytes in the lower chambers. Mesenchymal stem cell ultrastructural changes occurred, including a larger euchromatin nucleus, when the cells were placed in medium containing 10 percent fetal bovine serum, whereas basic fibroblast growth factor maintained the immature cell morphology for 4 days. Monolayer coculture also showed human mesenchymal stem cell changes in ultrastructural morphology in the vicinity of the epidermal keratinocytes. These data suggest that human mesenchymal stem cells may interact with human epidermal keratinocytes to accelerate wound healing and coverage.

Ectopic bone formation facilitated by human mesenchymal stem cells and osteogenic cytokines via nutrient vessel injection in a nude rat model.

In vivo studies using bone marrow-derived mesenchymal stem cells are still uncommon. Applications for bone defect replacement in undesirable clinical circumstances such as large defects, bacterial or other pathogen-contaminated fields, and irradiated surgical wound bed necessitate vascularized bone regeneration. Use of a fascial flap including regenerated bone would be a very powerful tool for treatment. It would be especially beneficial in cases where normal bone regeneration is not expected due to a lack of sufficient blood supply, extensive surgical scarring, or bacterial contamination. In this study, we used nude rats in which the superficial epigastric flap of the experimental group was used to wrap around a mixture of human mesenchymal stem cells, bone morphogenetic protein-2, and basic fibroblast growth factor cytokines in a gelatin carrier. These rats showed significantly higher bone mineral density at 4 weeks compared to the other experimental groups containing phosphate buffered saline, human mesenchymal stem cells alone, or the two cytokines alone (p < 0.01). There were no remarkable histologic differences up to 7 days. At 2 weeks, more progressive vascularity and perivascular tissue deposits were seen in the experimental group. Basophilic mineral structure surrounded the fibroblast-like mesenchymal stem cells at 4 weeks, presumably osteoblastic or osteoclastic cell lining. Bone marker immunohistochemistry against alkaline phosphatase and osteocalcin revealed diffuse and distinct immunoreactivity in osteoblastic cells in the experimental group at 4 weeks. Further transcriptional expression of polyomavirus enhancer binding protein 2alphaA suggested that the human transplanted cells proceeded to osteogenic lineage in 4 weeks. These results may be useful as a new approach for bone regeneration.

A novel molecular marker of pituitary tumor transforming gene involves in a rat liver regeneration.

BACKGROUND: Pituitary tumor transforming gene (PTTG), homologous to a mammalian securin, plays a pivotal role in cell transformation, however, its biological function(s) in normal tissues is not fully understood. Because the liver is a regenerative organ, the relevant biological function of PTTG in the liver would be more feasible to understand PTTG. Also, PTTG may be involved in the liver regeneration. MATERIALS AND METHODS: Expressions of rat hepatic PTTG messengerRNA (mRNA) and cellular immunoreactivities during the cell proliferative period of the liver regeneration both in vitro and in vivo were tested. RESULTS: PTTG expression of the rat primary hepatocyte was stimulated by HGF in a dose dependent manner, and was suppressed when hepatocyte proliferation was inhibited by transforming growth factor-beta1. A positive PTTG immunoreactive co-localizing with 5-bromo-2'-deoxyuridine (BrdU) in the hepatocyte nucleus was found and there was a concurrent sister chromatin itself by the immunofluorescent labeling of PTTG with cytokeratin 18 (CK18). DISCUSSION: Since the correlation of PTTG mRNA expression, cell proliferation and immunoreactivity were observed in primary rat cultured hepatocytes, PTTG may be a novel marker of cell proliferation both in vitro and in vivo liver regeneration.

Leukemia inhibitory factor-transfected embryonic fibroblasts and vascular endothelial growth factor successfully improve the skin substitute wound healing by increasing angiogenesis and matrix production.

BACKGROUND AND OBJECTIVE: The combined application of cytokines on embryonic fibroblasts and dermal substitute were studied for optimal skin defect coverage. The mechanism of combined treatment of leukemia inhibitory factor (LIF)-transfected embryonic fibroblasts and vascular endothelial growth factor (VEGF) were elucidated and subsequently the in vivo applications of both were tested in an artificial dermal substitute. METHODS: Mouse embryonic fibroblast cells, BALB-3T3, were stably transfected with mouse full-length LIF cDNA and added to various doses of VEGF for detection of signaling interaction. LIF-transfected cells and VEGF treatment were tested with pig-tendon derived collagen dermal substitute in the backs of BALB/c male mice up to for 14 days. RESULTS: LIF-transfected cells as well as vector-transfected fibroblasts significantly proliferated by 1, 10, or 100 ng VEGF on days 3 and 5. Erk mitogen-activated protein (MAP) kinase phosphorylation was observed from 1 to 30 min in LIF-transfected and 10 ng of VEFG, and 1 to 60 min in LIF-transfected and 100 ng VEFG treatments. The cellular fibronectin levels also increased in LIF-transfected cells with 10 and 100 ng VEGF additions. In in vivo analyses, LIF-transfected embryonic fibroblasts with 50 microg of VEGF markedly enhanced collagen I expression and CD34 angiogenic marker on days 7 and 14. CONCLUSION: LIF transfection and VEGF treatment enhanced phosphorylated-Erk-MAP kinase in vitro. In vivo study revealed that the combined application of LIF transfection of embryonic fibroblasts with an angiogenic factor such as VEGF in the template of a dermal substitute induced greater skin collagen production and angiogenesis in the dermal substitute.

Cranial bone defect healing is accelerated by mesenchymal stem cells induced by coadministration of bone morphogenetic protein-2 and basic fibroblast growth factor.

To facilitate bone healing in difficult circumstances, and to replace conventional therapeutic modalities, highly purified bone marrow-derived human mesenchymal stem cells (hMSCs) were investigated for induction of their osteogenic lineage upon provision of cytokine cues in vitro and in the cranial defect model in vivo. Alkaline phosphatase-expressing cells were most frequently observed when the hMSCs were treated with 2.5 ng/ml of basic fibroblast growth factor (bFGF) and 50 ng/ml of bone morphogenetic protein (BMP)-2 for 4 days in culture after a 6-day incubation in osteogenic medium containing dexamethasone, ascorbic acid-2-phosphate, and beta-glycerophosphate. Four-millimeter full-thickness cranial defect wounds were made in male nude rats (F344/NJCl-rnu), whose deficit in the T cell compartment prevented T-cell-mediated cellular rejection. The animals were treated for 4 weeks with hMSCs and application of 10 microg each of bFGF and BMP-2 that had been soaked into a gelatin sponge carrier. Significant bone mineral density was observed by dual X-ray absorptiometry and this treatment also produced histologically mature osteocytes surrounded by both osteoblasts and osteoclasts expressing alkaline phosphatase and osteocalcin. The bone mineral densities and histological structures were matched at 8 weeks post-transplantation. Therefore, human bone marrow-derived mesenchymal stem cells are able to differentiate into an osteogenic lineage upon cytokine stimulation and accelerate healing in a nude rat cranial bone healing model.

Bone morphogenetic protein-2 regulates proliferation of human mesenchymal stem cells.

Human mesenchymal stem cells obtained from the iliac crest of a single donor were investigated for cell proliferation, cell cycle profile, gene expression, and ultrastructural changes using electron microscopy. The human mesenchymal stem cells significantly increased their cell number by day 2 after treatment with bone morphogenetic protein-2 alone, or basic fibroblast growth factor alone or combinations of both proteins under serum-free conditions (p < 0.01). The human mesenchymal stem cells showed marked expression of cell nuclear antigen, notably at day 1, and pituitary tumor transforming gene throughout the experiment, suggesting cell cycle progression by bone morphogenetic protein-2 treatment. In addition, strong cellular nuclear bromodeoxyuridine incorporation was seen by immunocytochemistry. Fluorescence-activated cell sorting also showed a similar pattern of cell cycle progression with bone morphogenetic protein-2 treatment in serum-free medium and 10% fetal bovine serum treatment. The bone morphogenetic protein-2-treated human mesenchymal stem cells showed heterochromatin in the nucleus, suggesting cell differentiation, and well-developed granular endoplasmic reticulum, indicative of protein production. Overall, the human mesenchymal stem cells successfully proliferated with appropriate cell cycle progression and the cell ultrastructural morphology suggested marked nuclear and granular endoplasmic reticulum induction by bone morphogenetic protein-2 treatment in serum-free medium.