Effects of locally applied adipose tissue-derived microvascular fragments by thermoresponsive hydrogel on bone healing.

Insufficient vascularization is a major cause for the development of non-unions. To overcome this problem, adipose tissue-derived microvascular fragments (MVF) may serve as vascularization units. However, their application into bone defects needs a carrier system. Herein, we analyzed whether this is achieved by a thermoresponsive hydrogel (TRH). MVF were isolated from CD-1 mice and cultivated after incorporation into TRH, while non-incorporated MVF served as controls. Viability of MVF was assessed immunohistochemically over a 7-day period. Moreover, osteotomies were induced in femurs of CD-1 mice. The osteotomy gaps were filled with MVF-loaded TRH (TRH + MVF), unloaded TRH (TRH) or no material (control). Bone healing was evaluated 14 and 35 days postoperatively. MVF incorporated into TRH exhibited less apoptotic cells and showed a stable vessel morphology compared to controls. Micro-computed tomography revealed a reduced bone volume in TRH + MVF femurs. Histomorphometry showed less bone and more fibrous tissue after 35 days in TRH + MVF femurs compared to controls. Accordingly, TRH + MVF femurs exhibited a lower osseous bridging score and a reduced bending stiffness. Histology and Western blot analysis revealed an increased vascularization and CD31 expression, whereas vascular endothelial growth factor (VEGF) expression was reduced in TRH + MVF femurs. Furthermore, the callus of TRH + MVF femurs showed increased receptor activator of NF-κB ligand expression and higher numbers of osteoclasts. These findings indicate that TRH is an appropriate carrier system for MVF. Application of TRH + MVF increases the vascularization of bone defects. However, this impairs bone healing, most likely due to lower VEGF expression during the early course of bone healing. STATEMENT OF SIGNIFICANCE: In the present study we analyzed for the first time the in vivo performance of a thermoresponsive hydrogel (TRH) as a delivery system for bioactive microvascular fragments (MVF). We found that TRH represents an appropriate carrier for MVF as vascularization units and maintains their viability. Application of MVF-loaded TRH impaired bone formation in an established murine model of bone healing, although vascularization was improved. This unexpected outcome was most likely due to a reduced VEGF expression in the early phase bone healing.

Primary T cells for mRNA-mediated immunotoxin delivery.

Immune cells become increasingly attractive as delivery system for immunotoxins in cancer therapy to reduce the intrinsic toxicity and severe side effects of chimeric protein toxins. In this study, we investigated the potential of human primary T cells to deliver a secreted immunotoxin through transient messenger RNA (mRNA) transfection. The chimeric protein toxin was directed toward the neovasculature of cancer cells by fusing a truncated version of Pseudomonas exotoxin A (PE38) to human vascular endothelial growth factor (VEGF) and to the single chain variable fragment (scFv) of anti-Her2/neu. Protocols for the transient transfection of human embryonic kidney cells (HEK293) as well as activated primary human T cells were established. Transient transfection with mRNA coding for the immunotoxins e23-PE38, VEGF-PE38 and its attenuated variant VEGF-PE38D yielded efficient expression and secretion. Mass spectrometry analysis endorsed that a fraction of VEGF-PE38D was properly translocated into the endoplasmic reticulum. Furthermore, cytotoxic activity of immunotoxin secreting T cells toward cancer cells was confirmed in co-culture with ovarian adenocarcinoma cells in the presence of a bispecific antibody (bsAb), highlighting the potential of primary T cells for mRNA-mediated immunotoxin delivery.

[Cataract surgery in patients with end-stage glaucoma]. / Katarakt-Operation bei Patienten mit Glaucoma fere absolutum.

BACKGROUND: The purpose of this study was to assess the benefit of cataract surgery in patients with advanced cataract and glaucoma. METHODS: In a prospective study, we investigated 12 consecutive patients (12 eyes). Inclusion criteria were the diagnosis of cataract and end-stage glaucoma with a cup-disc ratio (CD) of 0.9-1.0 and marked visual field defects with partially preserved central function. Preoperatively, at the third postoperative day and after 6 months (2-11 months), the visual acuity (V), the intraocular pressure (IOP), the number of antiglaucomatous drugs and the visual fields were assessed. Furthermore, the surgical procedure and possible complications were noted. In all patients cataract surgery was performed with topical anaesthesia. RESULTS: 10 patients were treated with cataract surgery alone, whereas 2 patients underwent combined cataract and glaucoma surgery. The mean visual acuity improved significantly from 0.3 to 0.5 (p = 0.007). Additionally a significant intraocular pressure reduction of 4.4 mm Hg (p = 0.007) was observed. The number of antiglaucomatous drugs decreased from 1.5 preoperatively to 0.8 postoperatively. The mean deviation (MD) improved from -27.5 dB up to -26.4 dB (p = 0.036) after 6 months. CONCLUSION: Patients with progressive cataract and end-stage glaucoma can benefit from cataract surgery. Although marked visual field defects were present, an increase in visual acuity as well as a decrease of intraocular pressure may be achieved without worsening of the visual fields.

[Evaluation of eye patching after cataract surgery in topical anesthesia]. / Zur Notwendigkeit eines Augenverbandes nach Kataraktoperation in Tropfanästhesie.

BACKGROUND: Although postoperative eye patching is a common practice its background is not well known. Therefore the necessity of eye patching after cataract surgery in topical anesthesia from the medical point of view and the patients' subjective opinion was studied. PATIENTS AND METHODS: In this prospective and randomized study 133 patients received after cataract surgery either no covering of the eye (group1), a transparent eye shield for four hours (group 2), an eye pad for four hours (group 3) or an eye pad until the next morning (group 4). Clinical findings were noted and local symptoms, such as pain, foreign body sensation, tearing and photophobia were documented on a visual analogue scale (0 - 10). Furthermore, a questionnaire concerning the subjective opinion was handed out to the patient. RESULTS: The clinical findings revealed no significant differences between the groups. The mean values for local pain were 0.94 +/- 1.56, for the foreign body sensation 1.41 +/- 2.02, for tearing 0.99 +/- 1.8 and for photophobia 1.05 +/- 1.99. Comparing the groups there was significantly more pain and foreign body sensation reported by the patients in group 3, who received eye patching for 4 hours. 91 % of the unpatched patients had no discomfort, whereas 53 % of the patients wearing an eye pad until the next morning considered it as unnecessary. CONCLUSION: After cataract surgery in topical anesthesia only mild symptoms were noted. There were no significant differences between the groups in the objective clinical findings and the subjective feeling. These results indicate that after cataract surgery eye patching could be unnecessary.