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We designed long-term in vivo experiments to study rabbit urethral regeneration and remodelling over a hyaluronan biodegradable prosthesis. Seven months after the resection of a 1.5-cm-long tract of the urethra and its substitution with the prosthesis, radiological analysis showed the disappearance of the implant and the re-establishment of urethral continuity along the transmural defect. The regenerated tissue remodelled around the implant and exhibited good distensibility under pressure. Histological evaluation showed that the neo-urethra was lined with transitional epithelium and the stroma contained abundant elastic fibres. An examination of the pattern of the major cytoskeletal and cytocontractile proteins of smooth muscle cells and fibroblasts was able to distinguish fibroblasts from smooth muscle cells and myofibroblasts in the neo-urethra. These experiments provide evidence for the potential, successful use of biocompatible/bioresorbable devices for reconstructive surgery of the urethra.

Aim Thrombosis secondary to renal closed abdominal trauma is a rare event, most of the time it is clinically silent. We report here our experience. Materials and Methods. This is the case of a boy came to our observation after a road trauma with motorbike fall-out. The boy arrived in ED for head injury. The patient, stable for haemodynamics, had lacerated and contused injuries at pelvis and right buttock level. He underwent chest x-rays, brain CT and neurosurgery examination: all resulted negative. There was no macrohematuria, nor lumbar pain. Objectively abdomen was treatable. The patient was referred to temporary observation for 12 hours when he was asked to undergo abdomen ultrasound, which showed no documented lesions except for fluid collection at the pelvic level. To rule out all doubts, the patient had an abdominal CT scan, which showed a silent left kidney with suspected thrombosis at left renal level. The patient was sent to our attention after 15 hours: we decided to perform immediately selective arteriography with thrombus lysis. The arteriography documented a massive thrombosis. The thrombus lysis was impossible to be performed. To maintain the perfect functionality of the contralateral kidney we decided not to proceed further, but to perform only left nephrectomy. During surgery mesocolon laceration occurred, so the patient underwent also colic resection. Discussion. Thrombosis secondary to a closed renal abdominal trauma is an uncommon event, with little clinical expression. It is the consequence of an injury. Deceleration produces arterial dissection, which alters the blood flow to the kidney, which is then twisted and complicated with renal thrombosis. Quite common is the association with diaphragmatic rupture or urethral detachment. The alterations of renal parenchyma in the early hours are detectable only through CT scan, which represents the method of election, and which can highlight a functionally silent kidney. Conclusions. Renal thrombosis requires that diagnosis is done within the first 12 hours; a rapid revascularization should be promptly attempted.

Purpose: We evaluate the results of an elective cardiopulmonary bypass conceived to minimize the surgical risk related to its use with temporary circulatory arrest and deep hypothermia in the treatment of patients with renal tumor extending into the right atrium.Materials and Methods: From July 1996 to December 2000, 19 patients with renal neoplasm and venous involvement were admitted to our department. Three patients 4, 57 and 58 years old with a right (2) and left (1) renal tumor extending into the right atrium underwent radical nephrectomy and tumor thrombus removal using a normothermic cardiopulmonary bypass. The bypass circuit was connected with a vacuum assisted venous drainage giving a negative pressure of 20 to 40 mm. Hg. Neither circulatory arrest nor hypothermia was used. Tumor thrombus was extracted through a longitudinal “cavotomy” and removed along with the kidney.Results: Total cardiopulmonary bypass time was 14, 19 and 22 minutes, respectively. No intraoperative or postoperative com...

— Intracavitary chemo or immunotherapy for upper urinary tract transitional cell carcinomas has been recently introduced. From published data, Bacillus Calmette-Guerin appears to give significant and durable response rates (70% as prophylaxis, 81% as therapy) with acceptable side effects. The anecdotal use of chemotherapeutical agents prevents any conclusion being made about the therapeutical impact. To date, intracavitary percutaneous Bacillus Calmette-Guerin treatment is suggested for patients with “in situ” or occult carcinoma of the upper urinary tract, bilateral and synchronous tumors and/or with severe renal failure or not suitable for radical surgery.

AIMS--The objective of this study was to investigate alterations in conjunctival collagen and proteoglycans in the conjunctival giant papillae of patients with vernal keratoconjunctivitis (VKC). METHODS--Tissue samples from tarsal giant papillae of seven eyes from five patients with VKC, and five tarsal conjunctival samples from five normal patients were obtained. Tissues were processed and stained with haematoxylin and eosin, Van Gieson, trichromic Mallory, toluidine blue, Alcian blue, and alkaline Giemsa. Collagen extraction was performed in acetic acid and pepsin, total collagen was quantified using hydroxy-proline levels, and collagen types I and III were analysed by gel electrophoresis (SDS-PAGE). Proteoglycans were quantified using uronic acid levels. RESULTS--Histological evaluation showed a significant increase of mast cells in the epithelium (0/mm2 v 147/mm2, p < 0.01) and in the stroma (5.1/mm2 v 80/mm2, p < 0.01) of VKC patients. Collagen fibres were thicker and arranged irregularly, with the total amount significantly increased. Owing to an increased percentage of type III collagen, the ratio of collagen types I to III was decreased. Proteoglycans were also reduced in VKC samples. CONCLUSION--The well known morphological abnormalities observed in VKC correspond to alterations in the ratio between collagens and proteoglycans, and between different types of collagen. The greatly increased number of mast cells found in these tissues suggests an active role for these cells in the abnormal connective tissue metabolism observed in VKC.

Several clinical trials have shown intravesical bacillus Calmette-Guerin (BCG) to be effective in the prophylaxis of papillary tumour recurrences and in the therapy of bladder carcinoma in situ (CIS), as well as in delaying progression to muscle invasion. Nevertheless, the optimal regimen of BCG therapy for superficial bladder cancer has still to be defined. In a previous phase II trial, a low-dose regimen (BCG Pasteur strain, 75 mg) was able to achieve clinically significant response rates with a decrease in side-effects compared with other reported studies using standard-dose BCG. However, a phase III randomized trial - low dose versus standard dose (BCG Pasteur strain, 75 vs. 150 mg) - was considered necessary to clarify definitively the relationships between dose, efficacy and toxicity. The results of the interim analysis of 183 patients (performed in 1993) have shown response rates to be better in patients submitted to a low-dose BCG regimen (p=0.0009) and a significant decrease in most of the common side-effects (cystitis, fever, haematuria; p

Thrombosis secondary to renal closed abdominal trauma is a rare event, most of the time it is clinically silent. We report here our experience. MATERIALS AND METHODS. This is the case of a boy came to our observation after a road trauma with motorbike fall-out. The boy arrived in ED for head injury. The patient, stable for haemodynamics, had lacerated and contused injuries at pelvis and right buttock level. He underwent chest x-rays, brain CT and neurosurgery examination: all resulted negative. There was no macrohematuria, nor lumbar pain. Objectively abdomen was treatable. The patient was referred to temporary observation for 12 hours when he was asked to undergo abdomen ultrasound, which showed no documented lesions except for fluid collection at the pelvic level. To rule out all doubts, the patient had an abdominal CT scan, which showed a silent left kidney with suspected thrombosis at left renal level. The patient was sent to our attention after 15 hours: we decided to perform immediately selective arteriography with thrombus lysis. The arteriography documented a massive thrombosis. The thrombus lysis was impossible to be performed. To maintain the perfect functionality of the contralateral kidney we decided not to proceed further, but to perform only left nephrectomy. During surgery mesocolon laceration occurred, so the patient underwent also colic resection. DISCUSSION. Thrombosis secondary to a closed renal abdominal trauma is an uncommon event, with little clinical expression. It is the consequence of an injury. Deceleration produces arterial dissection, which alters the blood flow to the kidney, which is then twisted and complicated with renal thrombosis. Quite common is the association with diaphragmatic rupture or urethral detachment. The alterations of renal parenchyma in the early hours are detectable only through CT scan, which represents the method of election, and which can highlight a functionally silent kidney. CONCLUSIONS. Renal thrombosis requires that diagnosis is done within the first 12 hours; a rapid revascularization should be promptly attempted.

We evaluate the results of an elective cardiopulmonary bypass conceived to minimize the surgical risk related to its use with temporary circulatory arrest and deep hypothermia in the treatment of patients with renal tumor extending into the right atrium.From July 1996 to December 2000, 19 patients with renal neoplasm and venous involvement were admitted to our department. Three patients 4, 57 and 58 years old with a right (2) and left (1) renal tumor extending into the right atrium underwent radical nephrectomy and tumor thrombus removal using a normothermic cardiopulmonary bypass. The bypass circuit was connected with a vacuum assisted venous drainage giving a negative pressure of 20 to 40 mm. Hg. Neither circulatory arrest nor hypothermia was used. Tumor thrombus was extracted through a longitudinal "cavotomy" and removed along with the kidney.Total cardiopulmonary bypass time was 14, 19 and 22 minutes, respectively. No intraoperative or postoperative complications due to surgical technique occurred. No significant bleeding was observed at the time of cavotomy and all neoplastic tissue was removed. Pathological examination documented renal cell carcinoma in 2 cases and Wilms tumor in 1. All the patients are alive 30, 42 and 15 months, respectively, after the operation.Normothermic cardiopulmonary bypass with vacuum assisted venous drainage makes circulatory arrest and hypothermia unnecessary and avoids the potential complications associated with these procedures. With respect to veno-venous shunts this technique guarantees complete surgical control of the thrombus and avoids the need for extensive dissection of the retrohepatic vena cava and Pringle maneuver.

From January 1989 to September 1992, 5 patients (age 58–68 years) were treated with percutaneous Pasteur BCG for superficial transitional cell carcinoma of the upper urinary tract. In 4 cases BCG was prophylactic after endourological resection of the tumors. In one case of bilateral Tis of the upper urinary tract, BCG was administered for therapeutical purposes. Every patient received 375 mg Pasteur BCG in 250 ml saline, every 2 weeks for 6 consecutive weeks in 2 patients, 5 weeks in 1, and 4 in 2. No major complications were observed. After a median follow-up of 10.2 months, 4 patients are disease free, 1 patient underwent nephroureterectomy. Percutaneous BCG in upper tract superficial transitional cell cancer seems a feasible option in selected cases.

This paper presents a dermal replacement technique for cutaneous wounds of diverse origin. An autologous tissue-engineered dermal-like structure was implanted in one patient with an acute surgical excision of a nevus and in another patient with a chronic post-traumatic ulcer. The dermal constructs were well integrated with the wound area, and, at 3 weeks, an epidermal covering was implanted. In both cases, 3 to 8 weeks after autografting, good integration of the transplanted epidermis with the surrounding native skin and a good take over the dermal-like tissue occurred. The use of this technology, despite its high cost, is increasing. Clinicians choose it because it helps heal critical wounds for which current therapies have failed.

To study the extracellular composition of giant papillae in vernal keratoconjunctivitis (VKC) and the expression of growth factors that may stimulate fibrosis.Upper conjunctival specimens were obtained by biopsy in 9 patients affected by active tarsal VKC (14 eyes) and 10 normal control subjects. Immunohistochemistry was performed on tissue sections using monoclonal antibodies (mAbs) for collagens I, III, and VII; tumor necrosis factor (TNF)-alpha; transforming growth factor (TGF)-ss1; basic fibroblast growth factor (bFGF); and platelet-derived growth factor (PDGF). The mAbs anti-tryptase, anti-CD4, anti-CD68, and anti-EG2 were used as markers for mast cells, T-helper lymphocytes, macrophages, and eosinophils, respectively. Immunofluorescent double-staining for growth factors and cell markers was performed in VKC tissues.Immunostaining was highly positive for collagens I, III, and VII in the subepithelium of VKC conjunctiva. Image analysis showed a significant increase of staining per tissue area for both collagens I and VII and increased basal membrane length. The number of cells positive for TNF-alpha, TGF-ss, bFGF, or PDGF was significantly higher in VKC tissue than in control samples. Double staining showed that eosinophils and macrophages were the main sources of PDGF and that FGF was expressed by 46% of mast cells. Significant PDGF and FGF staining was observed in the conjunctival epithelium and vascular endothelium of all VKC tissues.In giant papillae of VKC, the extracellular matrix is characterized by overproduction of collagens. Expression of growth factors in the conjunctiva by resident cells (mast cells, epithelial cells, endothelial cells) and inflammatory cells (macrophages, eosinophils) may contribute to papillae formation and fibrosis evolution in chronic ocular allergic diseases.

Tissue engineering offers the possibility to reconstruct tissue substitutes in order to replace lost or damaged tissues. The availability of appropriate biomaterial devices is essential to allow in vitro cultured cells to behave as in the original tissues in vivo. In our studies we utilized a seminatural biomaterial made up by the benzyl ester of hyaluronan to grow keratinocytes, fibroblasts and chondrocytes. Keratinocytes and fibroblasts were isolated from human foreskin. Cells were separately cultured on two different hyaluronan based biomaterial devices for the first 15 days and then co-cultured for an additional period of 2 weeks. Keratinocytes gave rise to a well-differentiated epithelial layer, while fibroblasts were able to synthesize all the main extracellular molecules inside the biomaterial spaces, forming dermal-like tissues. When these two tissues were co-cultured, a skin equivalent was formed with a dermal-epidermal junction. Chondrocytes were obtained from chick-embryo sterna and cultured for 21 days inside a non-woven scaffolding made up of a hyaluronan-based biomaterial. Cells were able to organize themselves into nodules embedded in a dense metachromatic substance in which type II collagen was present. Data from this study suggest that this novel class of hyaluronan derived biomaterials is suitable for different cell culture and in vitro tissue reconstruction.

Erectile function of adult (8-month-old) and aged (27-month-old) rats was investigated by in vivo and in vitro assays. Reflexogenic tonic erections were evoked in vivo by electrostimulation of the dorsal nerve of the penis. Aged rats developed tendentially low intracavernosal pressures, and the kinetics of erection and detumescence were significantly lower than in adult animals. The erectile tissue isolated from aged rats exhibited poor response to papaverine. When precontracted with norepinephrine, aged tissues required a 3-fold increase of papaverine concentration to full relaxation. Functional measurements were coupled with morphological analysis of elastic fibres of the tunica albuginea. Light microscopy showed degenerative signs of elastic fibres of aged rat specimens. Taken together, the present findings show that physiological aging is associated with penile tissue stiffness and abnormal corporal compliance.

An established anterior cruciate ligament deficiency-induced articular cartilage degeneration was used to evaluate the effects of intrasynovial injection of hyaluronic acid upon cartilage destruction. In this study, proteoglycan solubility under associative and dissociative conditions was compared in two treatment protocols at intervals of seven, 13, and 17 weeks after surgical breakage of the anterior cruciate ligament in 2.5-year-old Beagle dogs. Untreated joints showed a marked increase in both total soluble glycosaminoglycan (GAG measured as uronic acid) and in the associative fraction. In both treated groups, there was a reduced amount of soluble GAG. Cessation of treatment after seven weeks caused gradual regression, with an increasing amount of CaCl2-soluble material in the associative fraction, while inception at seven weeks gave biochemical evidence of reversal, with increasing GAG present in the guanidine-soluble (dissociative) fraction on the insoluble residue. The protective effects of hyaluronic acid suggest the potential clinical application of this therapy in retarding the advance of osteoarthritis.

Circular dichroism measurements on aqueous solutions of alpha-elastin have given evidence of beta-bend structure at elevated concentrations. When Ca2+ was added, the concentration-dependent conformational transition was somewhat inhibited and the binding of the metal ion was shown, by means of equilibrium dialysis, to be essentially independent from alpha-elastin concentration in the range of 1 to 10 mg/ml. The results obtained are discussed in connection with the elasticity and calcification of elastin.

The effect of the intraarticular sodium hyaluronate (HA) injection on the osteoarthritic knee joint has been evaluated in dogs using an experimental model of osteoarthritis induced by sectioning the anterior cruciate ligament. Seven weeks after surgery, the damage, graded according to Mankin's scale, was significantly reduced in knee joints treated with HA from the second week postsurgery compared to untreated joints. When intraarticular HA therapy was initiated after the seventh week, osteoarthritis progression was still reduced compared to controls. Both morphology and morphometry showed a beneficial effect of HA on the cartilage response to the damage, as well as a clearcut inhibitory effect on the development of the fibroblastlike cell layer on the articular cartilage in untreated joints. The beneficial effects on the cartilage integrity and response to osteoarthritic damage might be related to a primary effect of HA on the cartilage surface. However, these effects do not exclude the possibility that, in addition, HA might act on the synovial membrane by limiting the synovial reaction.

Circular dichroism measurements on aqueous solutions of alpha-elastin have given evidence of beta-bend structure at elevated concentrations. When Ca2+ was added, the concentration-dependent conformational transition was somewhat inhibited and the binding of the metal ion was shown, by means of equilibrium dialysis, to be essentially independent from alpha-elastin concentration in the range of 1 to 10 mg/ml. The results obtained are discussed in connection with the elasticity and calcification of elastin.

Summary Free aldehyde-containing compounds have been isolated from the acidic hydrolysis of purified elastin. These substances gave bisulphite addition compounds, which could be retained on an AG 1-X 4 column and then selectively separated from the amino acids present. Peptides containing aldehyde groups have also been isolated from a mild alkaline hydrolysate of elastin. These peptides were retained on a QAE-Sephadex column that had previously been treated with H 2 S0 3 .

Using alcoholic KOH, elastin has been hydrolyzed and two homogeneous fragments have been isolated by Amberlite IR-120 chromatography. The peptides have been studied by circular dichroism in different solvents and in presence of Ca++ ions. The results are discussed from the standpoint of the free conformation in solution and the Ca++ ions complexation.

The present study describes the production of hyaluronan based porous microparticles by a semi-continuous gas anti-solvent (GAS) precipitation process to be used as a growth factor delivery system for in vivo treatment of ulcers. Operative process conditions, such as pressure, nozzle diameter and HYAFF11® solution concentrations, were adjusted to optimize particle production in terms of morphology and size. Scanning electron microscopy (SEM) and light scattering demonstrated that porous nano-structured particles with a size of 300 and 900 nm had a high specific surface suitable for absorption of growth factors from the aqueous environment within the polymeric matrix. Water acted as a plasticizer, enhancing growth factor absorption. Water contents within the HYAFF11® matrix were analyzed by differential scanning calorimetry (DSC). The absorption process was developed using fluorescence dyes and growth factors. Immunohistochemical analysis confirmed the high efficiency of absorption of growth factor and a mathematical model was generated to quantify and qualify the in vitro kinetics of growth factor release within the polymeric matrix. In vivo experiments were performed with the aim to optimize timed and focal release of PDGF to promote optimal tissue repair and regeneration of full-thickness wounds.

The regeneration of damaged organs requires that engineered tissues mature when implanted at sites of injury or disease. We have used new analytic techniques to determine the extent of tissue regeneration after treatment of knee injury patients with a novel cartilage tissue engineering therapy and the effect of pre-existing osteoarthritis on the regeneration process. We treated 23 patients, with a mean age of 35.6 years, presenting with knee articular cartilage defects 1.5 cm2 to 11.25 cm2 (mean, 5.0 cm2) in area. Nine of the patients had X-ray evidence of osteoarthritis. Chondrocytes were isolated from healthy cartilage removed at arthroscopy. The cells were cultured for 14 days, seeded onto esterified hyaluronic acid scaffolds (Hyalograft C), and grown for a further 14 days before implantation. A second-look biopsy was taken from each patient after 6 to 30 months (mean, 16 months). After standard histological analysis, uncut tissue was further analyzed using a newly developed biochemical protocol involving digestion with trypsin and specific, quantitative assays for type II collagen, type I collagen, and proteoglycan, as well as mature and immature collagen crosslinks. Cartilage regeneration was observed as early as 11 months after implantation and in 10 out of 23 patients. Tissue regeneration was found even when implants were placed in joints that had already progressed to osteoarthrosis. Cartilage injuries can be effectively repaired using tissue engineering, and osteoarthritis does not inhibit the regeneration process.