Can autologous myoblasts be used as a potential bulking agent?

OBJECTIVE: To investigate the behaviour of donor myoblasts at the vesico-ureteric junction (VUJ) and to evaluate their potential as an autologous bulking agent, as myoblast transplantation has been shown to regenerate damaged or degenerated tissue, and it was postulated that they could be used to treat vesico-ureteric reflux. MATERIALS AND METHODS: Muscle biopsies were obtained from the lower limb muscles of 10 pigs. The quality of the cells was evaluated by electrophysiological and immunohistochemical tests. The cell membranes of myoblasts were labelled with PKH26, a fluorescent dye. Six weeks after taking of the muscle biopsies all pigs underwent cell transplantation; 30 x 10(6) cells suspended in transplantation medium (in 1-mL syringes) were injected at the VUJ, into the proximal urethra and the rhabdosphincter. At the VUJ volumes of 1 mL were injected, whereas in the urethra and rhabdosphincter small cell depots (0.1 mL) were injected. All the pigs were killed 8 weeks later, and the myoblasts and newly formed myofibres were identified using fluorescence microscopy, with a histological evaluation and investigation of potential local inflammatory reaction. RESULTS: Two to three intact layers of autologous myoblasts were found in the outer aspects of the large cell depots in the VUJ. Immunohistochemistry further showed that the myoblasts were only viable at these outermost borders of the large bulking areas, whereas necrosis with red fluorescent cell detritus was visible in the remaining central aspects of the large bulk of cells. By contrast, cells survived and formed myotubes in the wall of the proximal urethra and the rhabdosphincter where the small cell depots had been injected. CONCLUSIONS: In small depots, transplanted autologous myoblasts can survive and differentiate into myofibres, while in a large bulk of cells the vast majority of cells become necrotic. The present results show that myoblasts cannot be used for augmentation of large volumes of tissue or as a bulking agent.

The role of oxygen termination of nanocrystalline diamond on immobilisation of BMP-2 and subsequent bone formation.

Medical implants are increasingly often inserted into bone of frail patients, who are advanced in years. Due to age, severe trauma or pathology-related bone changes, osseous healing at the implant site is frequently limited. We were able to demonstrate that coating of endosseous implants with nanocrystalline diamond (NCD) allows stable functionalization by means of physisorption with BMP-2. Strong physisorption was shown to be directly related to the unique properties of NCD, and BMP-2 in its active form interacted strongly when NCD was oxygen-terminated. The binding of the protein was monitored under physiological conditions by single molecule force spectroscopy, and the respective adsorption energies were further substantiated by force-field-calculations. Implant surfaces refined in such a manner yielded enhanced osseointegration in vivo, when inserted into sheep calvaria. Our results further suggest that this technical advancement can be readily applied in clinical therapies with regard to bone healing, since primary human mesenchymal stromal cells strongly activated the expression of osteogenic markers when being cultivated on NCD physisorbed with physiological amounts of BMP-2.

Signs of inflammation after sciatic nerve block in pigs.

Nerve stimulators are widely used to assist with peripheral nerve blocks but do not eliminate the risk of nerve injury. We evaluated the histologic findings 6 h after sciatic nerve block with bupivacaine in pigs. When a motor response was still obtained with a current <0.2 mA (n = 10), the postmortem microscopic evaluation revealed lymphocytes and granulocytes sub-, peri-, and intraneurally in 5 (50%) of 10 pigs. No signs of inflammation were observed when the muscle contraction was achieved with a current between 0.3 and 0.5 mA (P = 0.03). In conclusion, the current required to elicit a motor response, the position of the needle tip, and the subsequent likelihood of nerve damage merit further evaluation.

Gemcitabine with cyclosporine or with tacrolimus exerts a synergistic effect and induces tolerance in the rat.

BACKGROUND: This study investigated the effect of the antineoplastic agent gemcitabine (dFdC) in combination with cyclosporine (CsA) or with FK506 on acute heart allograft rejection in a rat model. METHODS: Transplantations were performed in the fully allogeneic Lewis-to-Brown Norway strain combination. dFdC, CsA, and FK506 single-drug therapy and combinations of dFdC with CsA and FK506 were administered at various dosages starting on day 1 to prevent and on day 4 to treat acute rejection until day 20. Animals who did not reject their graft were intraperitoneally injected with 108 splenic donor-type lymphocytes. In addition, Lewis and third-party skin grafts were transplanted to these animals. RESULTS: Mean graft survival times under CsA, FK506, and dFdC monotherapy were 18.3/63.7 days (1 mg/5 mg per kg), 41.7 days, and 24.7/38.7 days (100 microg/150 microg per kg), respectively. CsA and FK506 in combination with dFdC prolonged graft survival to more than 100 days (CsA) and more than 95.2 days (FK506). Graft survival after treatment of an ongoing rejection was 21.5/38.3 days for CsA (1 mg/5 mg per kg) and 17.7/59.2 days for dFdC (100 microg/150 microg per kg). The combination of CsA+dFdC prompted indefinite survival of five of six hearts. Lymphocyte inoculation did not induce graft rejection. Notably, none of the Lewis, but all third-party, skin grafts were rejected immediately. Histomorphologic analysis of grafted hearts, however, demonstrated typical features of chronic rejection. CONCLUSIONS: The combination of CsA and FK506 with low-dose dFdC exerts a synergistic effect in the prevention and treatment of acute allograft rejection in this model. Although chronic rejection could not be prevented, strain-specific tolerance was achieved. Therefore, combining standard immunosuppressants with dFdC is a novel, promising strategy for prevention and treatment of acute allograft rejection.

Diffuse mesenterial sclerosis: a characteristic feature of chronic small-bowel allograft rejection.

Chronic rejection is the major cause of late intestinal allograft dysfunction. The aim of this study was to analyze in detail the histopathological features of chronic rejection in the ACI-to-Lewis rat model of intestinal transplantation. Chronic rejection was achieved in orthotopic small-bowel allografts (ACI-Lewis) by limited immunosuppression with cyclosporin A (CyA). Isogeneic transplants (ACI-ACI) as well as native bowels (ACI) with and without immunosuppression served as controls. Bowels were removed together with the mesenteries 90 days postoperatively and analyzed using sections stained with hematoxylin and eosin as well as Masson's trichrome. The slides were coded, randomized and analyzed by grading of histological abnormalities. The most striking alterations of the allografts were noticed in the mesenteries exhibiting an extensive infiltration by mononuclear cells accompanied by a progressive diffuse fibrosis with shrinking of the mesenteries. These changes were most pronounced in the perivascular areas of the mesenteric arteriae and venae rectae. Three of five allografts showed vasculitis with myointimal proliferation of the arteriae rectae. Focally, there was spill-over of the inflammatory cells onto the intestinal muscularis propria. The mucosa of the allografts showed mild blunting, lymphocytic infiltration of the crypt epithelium and increased crypt cell apoptoses. The submucosa was unaffected, and there were no detectable abnormalities of the enteric ganglion cells. The present data support the view that chronic rejection of intestinal allografts is characterized by a diffuse sclerosing mesenteritis which may significantly contribute to late graft dysfunction. The present model may be useful to study the pathomechanisms of this inflammatory fibrosing process.

Myoelectric activity during chronic small bowel allograft rejection in rats.

The aim of this study was to analyze the effect of chronic rejection on myoelectric activity of orthotopic small intestine transplants in vivo. Chronic rejection was achieved in orthotopic small bowel allografts (ACI-Lewis) using cyclosporin A for limited immunosuppression. Isogeneic transplants (ACI-ACI) as well as native bowels (ACI) with and without immunosuppression served as controls. Myoelectric activities were recorded after a 12-hr fasting period and analyzed visually. Pacemaker frequencies within grafts were significantly below normal values (group 1: 37; group 2: 31.7; group 3: 37; group 4: 32.3). MMC periods and MMC phases did not show significant differences. The propagation velocity of phase III contractions was elevated in the ileum of grafts undergoing chronic rejection (group 1: 1.79; group 2: 1.59; group 3: 2; group 4: 2.5). In conclusion, fasting motility is altered but preserved in intestinal allografts undergoing chronic rejection. Intrinsic reinnervation from the native duodenum, which is necessary to generate normal pacemaker frequencies, was observed neither in allografts nor in isografts after a three-month period.

Estradiol enhances murine cardiac allograft rejection under cyclosporin and can be antagonized by the antiestrogen tamoxifen.

BACKGROUND: An increased incidence of acute rejection episodes in female heart transplant recipients has been reported in experimental and clinical studies. However, the exact mechanisms of gender-specific differences in alloreactivity are not completely understood. METHODS: C57BL/10 (H-2b) hearts were transplanted into C3H/He (H-2 k) recipients. Four gender combinations were used to test the influence of donor and recipient sex on graft survival. Recipients were treated with CsA, 17beta-estradiol and/or tamoxifen. Additionally mice were ovariectomized prior to transplantation. RESULTS: Treated with CsA, allograft survival in female recipients was 9.16+/-0.41 days as compared with 15.16+/-1.72 days in males. Estradiol administration and oophorectomy had a significant impact on allograft survival in male and female mice under CsA treatment. Tamoxifen combined with CsA significantly prolonged graft survival in female recipients (13.16+/-1.16 days) as compared with CsA treatment alone (9.16+/-0.41 days). CONCLUSION: Female mice show earlier rejection episodes and a shorter graft survival than males. For the first time, tamoxifen has been shown to have a beneficial effect on heart allograft survival in female recipients.

Duodenal histology for monitoring treatment of acute rejection in pancreaticoduodenal allografts in rats.

BACKGROUND: Although the value of duodenal histology as a means to diagnose acute rejection in pancreaticoduodenal allografts has been validated, it is not known how the duodenum responds to antirejection treatment in comparison with the pancreas. METHODS: Diabetic Lewis rats received a pancreaticoduodenal allograft. Cyclosporine was given for 5 days and then discontinued for 2 days (group 1), for 4 days (group 2), for 6 days (group 3), for 8 days (group 4), for 9 days (group 5), and for 10 days (group 6). Two animals of each group were killed for histology at the end of immunosuppressive-free intervals. In the remaining rats, rejection was treated with methylprednisolone on 3 consecutive days. Duodenal histology was compared with pancreatic morphology before and after treatment of rejection. RESULTS: Duodenal histology had a positive and negative predictive value of 100% for detection of acute rejection in the pancreatic portion of the graft. After antirejection treatment, duodenal morphology was however less accurate (positive predictive value, 96%; negative predictive value, 67%). The Spearman correlation coefficient (p) of duodenal and pancreatic rejection grades was higher before antirejection treatment (p=1.0) than thereafter (p=0.724). Considering interstitial and vascular changes separately, vascular rejection correlated to a higher extent than interstitial rejection between the two portions of the graft (p=0.725 vs. p=0.677). CONCLUSIONS: Duodenal histology accurately predicts the initial diagnosis of rejection of the pancreas. However, after treatment of acute rejection, duodenal morphology is more likely to recover from rejection than the pancreas. Awareness of this phenomenon might be important for the interpretation of duodenal follow-up biopsies.