Falk Wehrhan

Histomorphologische Strukturveränderungen von Kopf-Hals-Gefäßen nach prä- oder postoperativer Radiotherapie

Hintergrund: Patienten mit Plattenepithelkarzinomen im Kopf- und Halsbereich werden zumeist multimodal strahlen-, chemotherapeutisch und/oder chirurgisch behandelt. Die Erfolgsrate einer mikrovaskulären Hart- und Weichgeweberekonstruktion nach vorausgegangener Radiotherapie ist neben anderen Faktoren limitiert durch die strahleninduzierten Veränderungen der mikrochirurgisch genutzten Anschlussgefäße im Kopf- und Halsbereich. Ziel der Untersuchungen war es daher, das Ausmaß pathomorphologischer Gefäßveränderungen in Abhängigkeit von der Gesamtreferenzdosis und dem Intervall zwischen Radiotherapie und/oder Chemotherapie zu erfassen. Patienten und Methoden: Im Zeitraum von Oktover 1995 bis März 2002 wurden insgesamt 348 Patienten mit 356 freien mikrovaskulären Hart- und Weichgewebetransplantaten primär oder sekundär rekonstruiert. In Abhängigkeit von der strahlentherapeutischen Vorbehandlung wurde folgende Gruppeneinteilung vorgenommen: Gruppe 1 = Patienten (n = 27) mit ausschließlich chirurgischer Behandlung. Gruppe 2 = Patienten (n = 29) mit einer neoadjuvanten Radiochemotherapie (40–50 Gy; 800 mg/m2 5-Fluorouracil [5-FU] und 20 mg/g2 Cisplatin) und primärer Rekonstruktion. Grupp3 = Patienten (n = 20) mit vorausgegangener Radiotherapie (60–70 Gy) und sekundärer Rekonstruktion. 209 Gefäßproben der Anschluss- und Transplantatgefäße wurden qualitativ histologisch auf Veränderungen der Media und Intima sowie quantitativ histomorphometrisch (NIH-Image) auf eine anteilige Zu- oder Abnahme der Media, der Intima oder des Lumens am Gesamtquerschnitt des Gefäßes untersucht (ANOVA SPSS V9). Ergebnisse: Die Ergebnisse zeigten ein signifikantes Überwiegen von Gefäßveränderungen des Grade II (Hyalinose der Media und Intima und eine Intimaablösung, p = 0,009) an den Anschlussarterien in der Gruppe 3 (präoperative Radiotherapie mit 60–70 Gy). An Transplantatarterien (p = 0,127), Anschluss- (p = 0,43) und Transplantatvenen (p = 0,54) wurden keine signifikanten qualitativen Gefäßwandveränderungen zwischen einer vorausgegangenen Radiotherapie und keiner Bestrahlung gesehen. Bei der Analyse der Anschlussarterien war die Ratio Media/Gesamtfläche in Gruppe 3 (Median 0,51; IQR 0,10) signifikant kleiner als in Gruppe 1 (p = 0,02) (Median 0,61; IQR 0,29) und Gruppe 2 (p = 0,046) (Median 0,58; IQR 0,19). Ein signifikanter Unterschied der Ratio Lumen/Gesamtfläche zeigte sich zwischen Gruppe 1 und 3 (p = 0,01) (Gruppe 1: Median 0,24; IQR 0,15; Gruppe 2: Median 0,34; IQR 0,15; Gruppe 3; Median 0,40; IQR 0,18). Schlussfolgerung: Nach einer Vorbestrahlung mit 60–70 Gy wurden signifikante qualitative und quantitative Gefäßwandveränderungen an den Anschlussarterien, nicht aber an den Anschlussvenen, den Transplantatarterien oder den Transplantatvenen festgestellt. Im Gegensatz zeigte eine neoadjuvante Radiochemotherapie (40–50 Gy, 5-FU und Cisplatin) bei der primären vaskulären Rekonstruktion 1,5 Monate nach neoadjuvanter Vorbehandlung keine signifikanten histologischen Gefäßveränderungen der vorbestrahlten Anschlussgefäße.Background: Patients with squamous cell carcinomas of the oral cavity are being increasingly treated by multimodal interdisciplinary regimes using a combination of surgery, chemo- and radiotherapy. Inflammatory alterations of the vascular endothelium following preoperative radiotherapy frequently cause healing delays of free flaps in the irradiated graft bed. The aim of the study was to investigate quantitative and qualitative changes of irradiated neck recipient vessels and transplant vessels used for microsurgical anastomoses in free flaps in patients undergoing preoperative radiotherapy or radiochemotherapy. Patients and Methods: In 348 patients (October 1995–March 2002) receiving primarly or secondarly 356 microvascular hard- and soft tissue reconstruction, a total of 209 vessels were obtained from neck recipient vessels and transplant vessels during anastomosis. Three groups were analysed: group 1 (27 patients) treated with no radiotherapy or chemotherapy; group 2 (29 patients) treated with preoperative irradiation (60–70 Gy) and chemotherapy (800 mg/m2/day 5-FU and 20 mg/m2/day cisplatin) 1.5 months prior to surgery; group 3 (20 patients) treated with radiotherapy (60–70 Gy) (median interval 78.7 months; IQR: 31.3 months) prior to surgery. From each of the 209 vessel specimens, 3 sections were investigated histomorphometrically, qualitatively and quantitatively (ratio media area/total vessel area) by NIH-Image-digitized measurements. To evaluate these changes as a function of age, radiation dose and chemotherapy, a statistical analysis was performed using an analysis of covariance and 2tests (p > 0.05, SPSS V10). Results: In group 3, qualitative changes (intima dehiscence, hyalinosis) were found in recipient arteries significantly more frequently than in groups 1 and 2. For group 3 recipient arteries, histomorphometry revealed a significant decrease in the ratio media area/total vessel area (median 0.51, IQR 0.10) in comparison with groups 1 (p = 0.02) (median 0.61, IQR 0.29) and 2 (p = 0.046) (mdeian 0.58, IQR 0.19). No significant difference was found between the vessels of groups 1 and 2 (p = 0.48). There were no significant differences in transplant arteries and recipient or tansplant veins between the groups. Age and chemotherapy did not appear to have a significant influence on vessel changes in this study (p > 0.05). Conclusions: Following irradiation with 60–70 Gy, significant qualitative and quantitative histological changes to the recipient arteries, but not to the recipient veins, could be observed. In contrast, irradiation at a dose of 40–50 Gy and chemotherapy given at a median interval of 1.5 months prior to operation did not lead to significant histological changes to the recipient vessels.

Vestibuloplasty: Porcine Collagen Matrix Versus Free Gingival Graft: A Clinical and Histologic Study

BACKGROUND A free gingival graft (FGG) is currently the gold standard for augmenting small areas of keratinized mucosa. The porcine collagen matrix (CM) represents an alternative to autologous tissue harvesting. This study aims to compare the CM versus FGGs for augmenting keratinized peri-implant mucosa based on clinical and histologic evaluations. METHODS The study included 14 patients who underwent a vestibuloplasty with either a FGG from the palate (n = 7) or the CM (n = 7). An implant-fixed vestibular retention splint was inserted for 30 days. Follow-up examinations were performed at 4, 10, 30, and 90 days after surgery. Width of keratinized mucosa was measured in the region of each implant (days 10, 30, and 90). After 90 days, a biopsy was harvested for histologic and immunohistologic analyses. To characterize newly formed soft tissue, the authors stained for tissue-and differentiation-specific markers, cytokeratin (CK) 5/6, 13, and 14, to detect presence or absence of keratinization. RESULTS The groups showed similar healing, with increased peri-implant keratinized mucosa. The CM group had overall significantly shorter operation times than the FGG group. Both groups showed similar overall shrinkage (32.98% CM versus 28.35% FGG). All biopsies showed a multilayered, keratinized, squamous epithelium. CKs 5/6 and 14 were detected in the basal and suprabasal layers, and spots of CK 13 were detected in the suprabasal layer. CONCLUSIONS During the whole observation period, both groups showed comparable clinical and histologic outcomes. Within the limitations of the present study, CM seems to be a promising alternative for the regeneration of keratinized mucosa without tissue harvesting. Comparative long-term studies are needed to investigate changes over time.

Differential impairment of vascularization and angiogenesis in bisphosphonate-associated osteonecrosis of the jaw-related mucoperiosteal tissue

OBJECTIVES Impaired vascularization in the etiopathology of aminobisphosphonate-associated osteonecrosis of the jaw (BONJ) is assumed, but evidence is lacking. This immunohistochemical study differentiated vascularization and angiogenesis in BONJ-adjacent mucoperiosteal tissue. STUDY DESIGN Twenty BONJ (after zoledronate treatment) and 20 control mucoperiosteal tissue samples were processed with an autostaining-based alkaline phosphatase-antialkaline phosphatase staining kit. Vascularization was assessed by CD31 staining and angiogenesis-related neovessels by CD105 staining. The ratio of stained capillary area to total area of visible field was assessed. Statistics included Bonferroni adjustment. RESULTS CD31-stained microvessels were detected in each section and CD105-stained neovessels in each control. BONJ-adjacent mucoperiosteal tissue showed significantly fewer CD105-positive vessels in capillary areas (P < .05) than control samples. CD31-stained capillary area was not significantly reduced in mucoperiosteal BONJ-samples. CONCLUSIONS Angiogenesis is impaired in BONJ-related mucoperiosteal tissue, but vascularization remains unaffected. Vessel remodeling and neovessel formation is delayed in BONJ, resulting in impaired tissue regeneration of bisphosphonate-exposed oral mucosa.

Transforming growth factor ß1 and ß2 (TGFβ2 / TGFβ2) profile changes in previously irradiated free flap beds†

Following preoperative radiotherapy prior to ablative surgery of squamous epithelial carcinomas of the head and neck region, inflammatory changes and the expression of cytokines involved in wound healing could be observed. These processes lead to a delayed healing of free flaps in the graft bed. The aim of the present experimental study was to analyze the expression profiles of transforming growth factor (activated TGFβ1, TGFβ2) and latency‐associated peptide (LAP) in the irradiated graft beds and the transition area between grafts and irradiated graft beds.

Small oral squamous cell carcinomas with nodal lymphogenic metastasis show increased infiltration of M2 polarized macrophages – An immunohistochemical analysis

BACKGROUND In solid malignancies the influence of immunological parameters - especially of macrophages - on invasiveness, metastatic potential and prognosis has been shown. There are no studies quantitatively analysing the macrophage polarization in oral squamous cell carcinoma (oscc). The aim of this study was to correlate macrophage polarization in the epithelial and stromal compartment of oscc with histopathologic parameters. METHODS T1 and T2 oscc samples (n = 34) were used. Automated immunohistochemical staining detected CD68, CD11c, CD163 and MRC1 positive cells. All samples were completely digitalized using whole slide imaging and the number of stained cells per area was assessed quantitatively. RESULTS Primary tumours with lymphogenic metastasis (N+) showed a significantly (p < 0.05) increased count of CD68, CD11c, CD163 and MRC1 positive cells in the epithelial fraction compared to N0 tumours. The ratio of CD163 positive cells (M2 macrophages) to CD68 positive cells (M1 and M2 macrophages) was significantly (p < 0.05) increased in N+ tumours. CONCLUSION An increased macrophage infiltration and an increased M2 polarization in primary oral squamous cell carcinomas with lymphogenic metastasis was shown. Macrophages that migrated into the epithelial tumour fraction seem to be of special biological importance. The results indicate a central role of macrophages in the progression of oscc.

Improved free vascular graft survival in an irradiated surgical site following topical application of rVEGF.

PURPOSE Wound healing disorders following surgery in preirradiated tissue are clinically well known and may even become more crucial with the increasing use of neoadjuvant chemoradiation protocols. Both the expression of vascular endothelial growth factor (VEGF) and endoglin (CD105) play a key role in neovascularization and wound healing after soft tissue grafts in irradiated and nonirradiated tissue. Modulation of neovascularization through the application of recombinant VEGF (rVEGF) may be a therapeutic option to reduce wound healing disorders in irradiated tissue. An experimental in vivo model was used to study the possible role of rVEGF for reduction of wound healing disorders and the promotion of neovascularization. METHODS AND MATERIALS A free myocutaneous gracilis flap was transplanted from the groin into the neck region of Wistar rats (weight 300-500 g) with and without previous irradiation of the neck region with 40 Gy: Group 1 (n = 7) radiotherapy alone; Group 2 (n = 14) flap transplantation alone and rVEGF; Group 3 (n = 14) radiotherapy, transplantation, and rVEGF. Time interval between irradiation and grafting was 10 +/- 1 day. 1.0 micro g rVEGF/500 microL phosphate-buffered saline was applied s.c. intraoperatively and on Days 1 through 7. Neovascularization (CD105) and endogenous VEGF expression were analyzed by means of immunohistochemistry on Days 3, 5, 7, 14, and 28 postoperatively and quantified as labeling indices (LI). RESULTS After irradiation there was a continuous significant reduction of the cytoplasmic VEGF expression (MEAN LI: 0.018 +/- 0.048) compared with the nonirradiated control (mean LI: 0.042 +/- 0.006) (p < 0.001). VEGF expression after flap transplantation without irradiation after VEGF application was at a constantly higher level from Day 3 (mean LI: 0.044 +/- 0.01) to Day 28 postoperatively compared with the control group (Day 3, mean LI: 0.028 +/- 0.006) (p < 0.001). As an indication of increased neovascularization after the local application of rVEGF, a significantly increased expression of CD105 was found in the transition area and graft bed from Day 7 on (p < 0.001). After irradiation and grafting there was a significant overall increase in the VEGF- and CD105-expression throughout Day 28 after rVEGF in the transition area (p < 0.001). CONCLUSION Whereas irradiation alone led to a downregulation of the endogenous VEGF expression, rVEGF application resulted in an increased expression and in a CD105 associated neovascularization after soft tissue grafting in irradiated tissues. Application of rVEGF may enable modulation of wound healing by influencing neovascularization. This could indicate a possible clinical approach for reducing fibrosis and chronic wound healing disorders in irradiated tissues.

Increased malignancy of oral squamous cell carcinomas (oscc) is associated with macrophage polarization in regional lymph nodes – an immunohistochemical study

BackgroundIt is largely accepted that specific immunological parameters in solid malignancies are associated with patient’s prognosis. Recently a correlation of macrophage polarization with histomorphological parameters could also be shown in oral squamous cell carcinoma (oscc). The observed tumor derived peripheral immune tolerance could be associated with the macrophage polarization in regional tumor draining lymph nodes.So far there are no studies analyzing the macrophage polarization in cervical lymph nodes of oscc patients. In the present study we aimed to correlate macrophage polarization in different anatomical lymph node compartments of patients diagnosed with oscc with histopathologic parameters of the primary tumor (T-, N-, L-, V-, Pn-status, grading).MethodsTumor free (n = 37) and metastatic (n = 17) lymph nodes of T1 and T2 oscc patients were processed for immunohistochemistry to detect CD68, CD11c, CD163 and MRC1 positive cells. Samples were digitized using whole slide imaging and the number of cells expressing the aforementioned markers in the region of interest quantitatively analyzed.ResultsThe malignancy of the primary tumor (defined by T-, L-, Pn-status, grading) correlated with the lymph node macrophage polarization. L1 and Pn1 tumor cases displayed a significantly (p < 0.05) decreased M1 and increased M2 polarization in the sinus of the lymph nodes. G3 cases presented a significantly (p < 0.05) increased M2 polarization in the sinus compared to G2 cases. T2 tumors had significantly (p < 0.05) increased M2 polarization in the interfollicular zone of regional lymph nodes compared to T1 tumors. Metastatic and non-metastatic lymph nodes did not differ regarding their macrophage polarization.ConclusionsThe current study revealed for the first time an influence of oscc on the macrophage polarization in regional lymph nodes. Markers of malignant behavior in the primary tumor were associated with a shift of macrophage polarization in lymph nodes from the anti-tumoral M1 type to the tumor-promoting M2 type. As tumor free and metastatic lymph nodes did not differ in terms of their macrophage polarization pattern, there must be other factors influencing the location for lymph node metastasis formation.

Exogenous Modulation of TGF-β1 Influences TGF-βR-III-Associated Vascularization during Wound Healing in Irradiated Tissue

Background and Purpose:Following preoperative radiotherapy prior to ablative surgery of squamous epithelial cell carcinomas of the head and neck region, wound-healing disorders occur. Previous experimental studies showed altered expression of transforming growth factor-(TGF-)β isoforms following surgery in irradiated graft beds. Altered levels of TGF-β1 are reported to promote fibrosis and to suppress vascularization during wound healing, whereas expression of TGF-β receptor-III (TGF-βR-III) is associated with vascularization. The aim of the study was to analyze the influence of anti-TGF-β1 treatment on TGF-βR-III-associated vascularization in the transition area between irradiated graft bed and graft.Material and Methods:Wistar rats (male, weight 300–500 g) underwent preoperative irradiation of the head and neck region with 40 Gy (four fractions of 10 Gy each; n = 16 animals). A free myocutaneous gracilis flap taken from the groin was then transplanted to the neck in all rats. The time interval between operation and transplantation was 4 weeks. Eight animals received 1 µg anti-TGF-β1 into the graft bed by intradermal injection on days 1–7 after surgery. On days 3, 7, 14, 28, 56, and 120, skin samples were taken from the transition area between transplant and graft bed and from the graft bed itself. Immunohistochemistry was performed using the ABC-POX method to analyze the TGF-βR-III and E-selectin expression. Histomorphometry was performed to analyze the percentage and the area of positively stained vessels.Results:A significantly higher expression of TGF-βR-III was seen in the irradiated and anti-TGF-β1-treated graft bed in comparison to the group receiving preoperative irradiation followed by transplantation alone. The percentage of TGF-βR-III positively staining capillaries from the total amount of capillaries in the anti-TGF-β1-treated graft bed was higher than in the group irradiated only. The total area of capillaries was also higher in the TGF-β1-treated group.Conclusion:Neutralizing of TGF-β1 activity in irradiated tissue undergoing surgery leads to a higher expression of TGF-βR-III and increased vascularization. TGF-βR-III seems to be associated with newly formed blood vessels during neovascularization in wound healing.Hintergrund und Ziel:Nach präoperativer Strahlentherapie und Chirurgie zur Behandlung von Plattenepithelkarzinomen im Kopf-Hals-Bereich treten Wundheilungsstörungen auf. Entzündliche Veränderungen und Änderungen der Zytokinexpression führen zu verzögerter Heilung und Fibrosierung. Experimentelle Voruntersuchungen zeigten eine erhöhte Expression der „transforming growth factor“-(TGF-)β-Isoformen TGF-β1, 2 nach Chirurgie im vorbestrahlten Transplantatlager. Die erhöhte TGF-β1-Expression fördert die Fibrosierung und vermindert die Vaskularisation; der Rezeptor TGF-βR-III ist hingegen mit einer Neovaskularisation assoziiert. Ziel der Untersuchung war die Analyse des Einflusses einer Anti-TGF-β1-Antikörper-Behandlung auf die Ausprägung der TGF-βR-III-assoziierten Kapillarneubildung im Übergangsbereich zwischen bestrahltem Transplantatlager und Transplantat.Material und Methodik:Wistar-Ratten (männlich, 300–500 g) wurden mit einer Dosis von 4 × 10 Gy im Halsbereich präoperativ bestrahlt (16 Tiere). Ein freier myokutaner Grazilislappen wurde bei allen Tieren in der Leistengegend gehoben und in den Halsbereich transplantiert. Das Zeitintervall zwischen Bestrahlung und Operation betrug 4 Wochen. Acht Tiere erhielten an den postoperativen Tagen 1–7 eine intradermale Injektion von 1 µg Anti-TGF-β1 in das Transplantatlager. An den Tagen 3, 7, 14, 28, 56 und 120 nach Operation wurden Hautbiopsien aus dem Übergangsbereich zwischen Transplantat und Transplantatlager gewonnen und immunhistochemisch gefärbt (ABC-POX-Methode). Die Expression von TGF-βR-III und E-Selectin wurde evaluiert. Zur Analyse der relativen Kapillaranzahl und der relativen Kapillarfläche wurde eine Histomorphometrie durchgeführt.Ergebnisse:Eine signifikant erhöhte Expression von TGF-βR-III wurde in der präoperativ bestrahlten und Anti-TGF-β1-behandelten Gruppe im Vergleich zur ausschließlich vorbestrahlten Gruppe nachgewiesen. Der Anteil TGF-βR-III-positiver Kapillaren an der Gesamtkapillarfläche war in der Anti-TGF-β1-behandelten Gruppe größer. Die Vaskularisation (Gefäßanzahl je Fläche) war in der Anti-TGF-β1-behandelten Gruppe ebenfalls größer.Schlussfolgerung:Die postoperative Applikation von neutralisierenden Antikörpern gegen TGF-β1 im vorbestrahlten Gewebe führt zu einer erhöhten TGF-βR-III-Expression und zu einer vermehrten Vaskularisation. TGF-βR-III ist offenbar mit Gefäßneubildung im Verlauf der Wundheilung assoziiert.

PEG matrix enables cell-mediated local BMP-2 gene delivery and increased bone formation in a porcine critical size defect model of craniofacial bone regeneration

PURPOSE This study addressed the suitability of a polyethylene glycol (PEG) matrix as scaffold for cell-mediated local BMP-2 gene transfer in a calvarial critical size defect (CSD) model. MATERIALS AND METHODS PEG matrix (degradation time 10 days) and PEG membrane (degradation time 120 days) were used in the pig calvarial model. Cylindrical (1 × 1 cm) CSD (9 per animal; 20 animals) were filled with: (i) HA/TCP, covered by PEG membrane (group 1); (ii) HA/TCP, mixed with PEG matrix (group 2); and (iii) HA/TCP mixed with BMP-2 transfected osteoblasts and PEG matrix (group 3). BMP-2/4 gene transfer: liposomal in vitro transfection of BMP-2/V5-tag fusion-protein. Quantitative histomorphometry (toluidine blue staining) after 2, 4 and 12 weeks assessed bone formation. Semiquantitative immunohistochemistry estimated the expression of BMP-2 and V5-tag. RESULTS Group 3 showed significantly higher new bone formation than groups 1, 2 at 4 (P < 0.05) and 12 (P < 0.02) weeks. BMP-2-V5-tag was detected for 4 weeks. BMP-2 expression in group 3 was higher compared to all other groups after 2 and 4 (P < 0.02) weeks. CONCLUSIONS The PEG matrix serves as scaffold for cell-mediated BMP-2 gene delivery in guided bone regeneration facilitating cell survival and protein synthesis for at least 4 weeks. Local BMP-2 gene delivery by PEG matrix-embedded cells leads to increased bone formation during critical size defect regeneration.

Msx-1 is suppressed in bisphosphonate-exposed jaw bone analysis of bone turnover-related cell signalling after bisphosphonate treatment

OBJECTIVES Bone-destructive disease treatments include bisphosphonates and antibodies against receptor activator for nuclear factor κB ligand (aRANKL). Osteonecrosis of the jaw (ONJ) is a side-effect. Aetiopathology models failed to explain their restriction to the jaw. The osteoproliferative transcription factor Msx-1 is expressed constitutively only in mature jaw bone. Msx-1 expression might be impaired in bisphosphonate-related ONJ. This study compared the expression of Msx-1, Bone Morphogenetic Protein (BMP)-2 and RANKL, in ONJ-affected and healthy jaw bone. MATERIAL AND METHODS An automated immunohistochemistry-based alkaline phosphatase-anti-alkaline phosphatase method was used on ONJ-affected and healthy jaw bone samples (n = 20 each): cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed to quantitatively compare Msx-1, BMP-2, RANKL and GAPDH mRNA levels. RESULTS Labelling indices were significantly lower for Msx-1 (P < 0.03) and RANKL (P < 0.003) and significantly higher (P < 0.02) for BMP-2 in ONJ compared with healthy bone. Expression was sevenfold lower (P < 0.03) for Msx-1, 22-fold lower (P < 0.001) for RANKL and eightfold higher (P < 0.02) for BMP-2 in ONJ bone. CONCLUSIONS Msx-1, RANKL suppression and BMP-2 induction were consistent with the bisphosphonate-associated osteopetrosis and impaired bone remodelling in BP- and aRANKL-induced ONJ. Msx-1 suppression suggested a possible explanation of the exclusivity of ONJ in jaw bone. Functional analyses of Msx-1- RANKL interaction during bone remodelling should be performed in the future.