Controlled release of vascular endothelial growth factor by use of collagen hydrogels

In vivo profile of vascular endothelial growth factor (VEGF) release from collagen hydrogels was investigated comparing that of hydrogel degradation while angiogenesis induced by the released VEGF was assessed. Collagen sponges were chemically cross-linked with different amounts of glutaraldehyde for various time periods. When 125I-labeled collagen hydrogels incorporating VEGF were subcutaneously implanted into the back subcutis of mice, the hydrogel radioactivity decreased with time, the decrement profile depending on the cross-linking conditions. The radioactivity was retained for longer time periods as the glutaraldehyde concentration and cross-linking time increased. Implantation study of collagen hydrogels incorporating 125I-labeled VEGF revealed that the remaining VEGF radioactivity decreased with time and the retention period was prolonged with the decreased hydrogel biodegradation. The slower the hydrogel degradation, the longer the period of VEGF retention. The collagen hydrogel incorporating VEGF induced significant angiogenesis around the implanted hydrogel, in marked contrast to VEGF in the solution form and VEGF-free empty hydrogel. The retention period of angiogenesis became longer with a decrease of the in vivo degradation rate of hydrogels. It is possible that the slower degraded hydrogel achieves a longer period of VEGF release, resulting in prolonged angiogenetic effect. We concluded that in our hydrogel system, biologically-active VEGF was released as a result of in vivo degradation of the hydrogel.     

A novel EBNA-1 titration method and putative anti-EBNA-1 protein

A novel and rapid EBNA-1 titration method has been developed which uses immunoprecipitation of specific DNA-protein complexes with EBNA-1-positive serum. The method is more sensitive than the conventional immunofluorescence method and has potential value as a diagnostic reagent for clinical laboratories. TPA induction of putative anti-EBNA-1 protein of cellular origin is discussed, which may play a key role for the shift from latent to lytic replication of EBV.     

Adipose tissue engineering based on human preadipocytes combined with gelatin microspheres containing basic fibroblast growth factor

Gelatin microspheres containing basic fibroblast growth factor (bFGF) were prepared for the controlled release of bFGF. Co-implantation with the gelatin microspheres enabled preadipocytes to induce adipose tissue formation at the implanted site. Preadipocytes isolated from human fat tissue were suspended with the gelatin microspheres containing bFGF and incorporated into a collagen sponge of cell scaffold. Following subcutaneous implantation of the collagen sponge incorporating human preadipocytes, and gelatin microspheres containing 1 microg of bFGF into the back of nude mice, adipose tissue was formed at the implanted site of collagen sponge within 6 weeks postoperatively although the extent depended on the number of preadipocytes transplanted and the bFGF dose. The formation of adipose tissue was significant compared with the implantation of collagen sponge incorporating human preadipocytes and 1 microg of free bFGF. The area of adipose tissue newly formed was increased with the number of preadipocytes transplanted until to 1.0 x 10(5) cells/site and thereafter leveled off. The maximum area was observed at the bFGF dose of 1 microg/site. The area was significantly smaller at the bFGF dose of 0.5 microg/site or larger than 1 microg/site. Immunohistochemical examination indicated that the adipose tissue newly formed was composed of human matured adipocytes. No adipogenesis was observed at the implanted site of collagen sponge incorporating either gelatin microspheres containing bFGF or human preadipocytes and the mixed gelatin microspheres containing bFGF and human preadipocytes. We conclude that combination of gelatin microspheres containing bFGF and preadipocytes with the collagen sponge is essential to achieve tissue engineering of fat tissue.     

Collagenous matrices as release carriers of exogenous growth factors

We have investigated the use of natural and synthetic collagenous matrices as carriers of exogenous growth factors. A bladder acellular matrix (BAM) was processed from rat bladder and compared with sponge matrix of porcine type 1 collagen. The lyophilized matrices were rehydrated by the aqueous solutions of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), platelet derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), insulin like growth factor-1 (IGF-1) and heparin binding epidermal growth factor-like growth factor (HB-EGF), to obtain the matrix incorporating each growth factor. The rehydration method enabled the growth factor protein to distribute into the matrix homogeneously. In vivo release test in the mouse subcutis revealed that, the property of BAM for growth factor release was similar to that of collagen sponge. Among the growth factors examined, bFGF release was the most sustained, followed by HGF and PDGF-BB. bFGF released from the two matrices showed similar in vivo angiogenic activity at the mouse subcutis in a dose-dependent manner. These findings demonstrate that the collagenous matrices function as release carriers of growth factors. This feature is promising to create a scaffold, which has a nature to control the tissue regeneration actively.     

Prefabrication of vascularized bone graft using guided bone regeneration

This article describes the prefabrication of a vascularized bone graft composed of autologous particulate cancellous bone and marrow (PCBM), a vessel bundle, and a biodegradable membrane. The PCBM was placed around the saphenous vessel bundle of rats and rolled with a biodegradable membrane of L-lactide-epsilon-caprolactone copolymer to prepare the prefabricated vascularized bone graft (group A). As controls, combinations of PCBM and membrane (group B), vessel bundle and membrane (group C), and PCBM and vessel bundle (group D) were prepared. A radiographic study revealed radio-opacity in the implantation site of group A 1 week later, in contrast to the other groups. Newly formed bone in the membrane roll was histologically confirmed, and neomicrovasculature circulating from the vessel bundle through the newly formed bone tissue was observed. The increase in alkaline phosphatase activity and osteocalcin content was significant for the group A preparation compared with the other groups. We concluded that the combination of autologous PCBM, a vessel bundle, and a biodegradable membrane was promising in the prefabrication of vascularized bone with good blood circulation.     

CD40 ligand deficiency: treatment strategies and novel therapeutic perspectives

Introduction: CD40 ligand (CD40L) deficiency or X-linked Hyper-IgM syndrome is a severe primary immunodeficiency caused by mutations in the CD40L gene. Despite currently available treatments, CD40L-deficient patients remain susceptible to life-threatening infections and have poor long term survival.

Areas covered: Here, we discuss clinical and immunological characteristics of CD40L deficiency as well as current therapeutic strategies used for patient management. This review highlights that beyond B cell defects, patients’ susceptibility to opportunistic pathogens might be due to impaired T cell and innate immune responses. In this context, we discuss how better knowledge of CD40L function and regulation may result in the development of new treatments.

Expert opinion: Despite the introduction of hematopoietic stem-cell transplantation, immunoglobulin replacement, granulocyte colony-stimulating factor (G-CSF) administration, and prophylactic antibiotic therapies, life-threatening infections still cause high morbidity and mortality among CD40L-deficient patients. The reasons for this inadequate response to current therapies remains poorly understood, but recent reports suggest the involvement of CD40L–CD40 interaction in early stages of the innate immune system ontogeny. The development of novel gene therapeutic approaches and the use of redirected immunotherapies represent alternative treatment methods that could offer reduced morbidity and mortality rates for patients with CD40L deficiency.     

Phase II study of adjuvant chemotherapy with paclitaxel and nedaplatin for uterine cervical cancer with lymph node metastasis

The purpose of this phase II trial was to assess the efficacy and toxicity of paclitaxel and nedaplatin (TN) as the initial postoperative adjuvant chemotherapy for uterine cervical cancer with lymph node metastases (LNM). Patients with FIGO stage IB1‐IIA2 squamous cell carcinoma of the uterine cervix were enrolled. Histological confirmation of LNM was mandatory. Intravenous paclitaxel at 175 mg/m² and nedaplatin at 80 mg/m² were administered every 28‐day cycle, of which there were 5 cycles after radical hysterectomy. Sixty‐two patients were enrolled in the study from November 2011 to July 2015. Their median age was 48.5 years (range 28‐64). The median tumor diameter was 37 mm (5‐64). Overall, 30 patients (48.4%) had 1 metastatic lymph node, 11 (17.7%) had 2, 3 (4.8%) had 3, 5 (8.1%) had 4, and 13 (21.0%) had 5 or more. With a median follow‐up of 45.7 months (range 23.4‐69.5), the 2‐year relapse‐free survival and 2‐year overall survival rates were 79.0% (90% CI, 69.0%‐86.2%) and 93.5% (95% CI, 83.7%‐97.5%), respectively. Almost all adverse events were relatively mild. Grade 3‐4 adverse events (NCI‐CTC ver. 4.0) that occurred in 5% or more of patients were neutropenia (60.7%) and infection (6.6%). The proportion of patients who completed 5 cycles of treatment was 90.3%. Postoperative adjuvant chemotherapy with TN for cervical cancer with LNM was demonstrated to be an effective and feasible treatment. A phase III trial is warranted to compare this with concurrent chemoradiotherapy.     

Detection of bone invasion by gingival carcinoma of the mandible: a comparison of intraoral and panoramic radiography and computed tomography

OBJECTIVE: To assess the diagnostic accuracy of panoramic radiography (PR), panoramic radiography combined with intraoral radiography (PR+IR), and CT in detecting the supero-inferior extent of tumor invasion of the mandible by gingival carcinoma. METHOD: PR, PR+IR, and CT images of the mandible in 37 patients with gingival carcinoma were evaluated by five oral radiologists for the supero-inferior extent of bone invasion using ROC analysis. The mean ROC curve area (Az) of each observer for the different imaging modalities was analysed by nonparametric two-way ANOVA. P<0.05 was considered statistically significant. RESULTS: The mean Az for the detection of bone invasion were 0.88+/-0.03 for PR, 0.77+/-0.12 for PR+IR, and 0.87+/-0.03 for CT (P=0.0907). The mean Az for the detection of bone invasion beyond the alveolus was 0.89+/-0.07 for PR, 0.85+/-0.08 for PR+IR, and 0.83+/-0.06 for CT (P=0.5438). The mean Az for the detection of bone invasion beneath the mandibular canal were 0.94+/-0.04 for PR, 0.94+/-0.02 for PR+IR, and 0.91+/-0. 04 for CT (P=0.2466). No statistically significant differences were observed in Az between PR, PR+IR, and CT. CONCLUSION: We consider that PR+IR should be adopted as the initial imaging modality to determine the extent of supero-inferior invasion of the mandible in gingival carcinoma.     

Co-culture of extracellular matrix suppresses the cell death of rat pancreatic islets

The purpose of this study was to investigate the effect of culture microenvironment on the cell death of isolated rat pancreatic islets. After isolation by the conventional collagenase digestion technique, the islets were cultured in a hydrogel of collagen type I mixed with collagen type III, type IV, and laminin. Irrespective of the type of mixture, islet cell death was significantly suppressed by their co-culture with the collagen hydrogel mixtures, although no change in islet morphology was observed. Co-culture with the collagen mixtures had no influence on the expressed mRNA level of insulin, glucagon, and somatostatin of the islets, or the islet secretion of hepatocyte growth factor (HGF), interleukin (IL)-1alpha, and IL-1beta. These findings suggest that three-dimensional culture in the collagen hydrogel and the mixture of laminin is able to maintain the cell viability of islets.     

In situ tissue engineering of periodontal tissues by seeding with periodontal ligament-derived cells

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.