Establishment and effects of allograft and synthetic bone graft substitute treatment of a critical size metaphyseal bone defect model in the sheep femur.

Assessment of bone graft material efficacy is difficult in humans, since invasive methods like staged CT scans or biopsies are ethically unjustifiable. Therefore, we developed a novel large animal model for the verification of a potential transformation of synthetic bone graft substitutes into vital bone. The model combines multiple imaging methods with corresponding histology in standardized critical sized cancellous bone defect. Cylindrical bone voids (10 ml) were created in the medial femoral condyles of both hind legs (first surgery at right hind leg, second surgery 3 months later at left hind leg) in three merino-wool sheep and either (i) left empty, filled with (ii) cancellous allograft bone or (iii) a synthetic, gentamicin eluting bone graft substitute. All samples were analysed with radiographs, MRI, µCT, DEXA and histology after sacrifice at 6 months. Unfilled defects only showed ingrowth of fibrous tissue, whereas good integration of the cancellous graft was seen in the allograft group. The bone graft substitute showed centripetal biodegradation and new trabecular bone formation in the periphery of the void as early as 3 months. µCT gave excellent insight into the structural changes within the defects, particularly progressive allograft incorporation and the bone graft substitute biodegradation process. MRI completed the picture by clearly visualizing soft tissue ingrowth into unfilled bone voids and presence of fluid collections. Histology was essential for verification of trabecular bone and osteoid formation. Conventional radiographs and DEXA could not differentiate details of the ongoing transformation process. This model appears well suited for detailed in vivo and ex vivo evaluation of bone graft substitute behaviour within large bone defects.

Preclinical testing of drug delivery systems to bone.

Bone defects do not heal in 5-10% of the fractures. In order to enhance bone regeneration, drug delivery systems are needed. They comprise a scaffold with or without inducing factors and/or cells. To test these drug delivery systems before application in patients, they finally need to be tested in animal models. The choice of animal model depends on the main research question; is a functional or mechanistic evaluation needed? Furthermore, which type of bone defects are investigated: load-bearing (i.e. orthopedic) or non-load-bearing (i.e. craniomaxillofacial)? This determines the type of model and in which type of animal. The experiments need to be set-up using the 3R principle and must be reported following the ARRIVE guidelines.

Phototherapy with LED light modulates healing processes in an in vitro scratch-wound model using 3 different cell types.

BACKGROUND: An effective way of modulating wound healing processes, including proliferation and apoptosis, is low-level light therapy. Because of several disadvantages of lasers, light-emitting diodes (LEDs) could be more feasible light sources. OBJECTIVE: To evaluate and compare the effects of blue and red light from LEDs on different cell types in an in vitro scratch-wound model. METHODS: Monolayers of C2C12 myoblasts, NIH/3T3 fibroblasts, and BICR10 keratinocytes were injured by mechanical scraping. Cells were illuminated on 5 consecutive days for 10 minutes by LED at 470 or 630 nm. Effects of light on in vitro wound healing were evaluated by analyzing time to closure, proliferation, apoptosis, and necrosis rates. RESULTS: Illumination substantially affected cell viability and cell growth. Blue light strongly decreased proliferation and augmented apoptosis in all 3 cell types and increased necrosis rates in C2C12 and NIH/3T3 cells. In contrast, red light did not alter apoptosis in either cell type but promoted proliferation in all 3 cell types with significant effects in C2C12 and NIH/3T3 cells and shortened time to closure in all 3 cell types. CONCLUSION: Light-emitting diode light illumination could be a therapeutic option and positively affect wound healing processes. By choosing appropriate wavelengths, variable effects can be achieved.

Low-frequency pulsed electromagnetic fields significantly improve time of closure and proliferation of human tendon fibroblasts.

BACKGROUND: The promotion of the healing process following musculoskeletal injuries comprises growth factor signalling, migration, proliferation and apoptosis of cells. If these processes could be modulated, the healing of tendon tissue may be markedly enhanced. Here, we report the use of the Somagen™ device, which is certified for medical use according to European laws. It generates low-frequency pulsed electromagnetic fields that trigger effects of a nature that are yet to be determined. METHODS: A 1.5-cm wide, linear scrape was introduced into patellar tendon fibroblast cultures (N = 5 donors). Treatment was carried out every second day. The regimen was applied three times in total with 30 minutes comprising pulsed electromagnetic field packages with two fundamental frequencies (10 minutes of 33 Hz, 20 minutes of 7.8 Hz). Control cells remained untreated. All samples were analyzed for gap closure time, proliferation and apoptosis one week after induction of the scrape wound. RESULTS: The mean time for bridging the gap in the nontreated cells was 5.05 ± 0.33 days, and in treated cells, it took 3.35 ± 0.38 days (P <0.001). For cell cultures with scrape wounds, a mean value for BrdU incorporation of OD = 0.70 ± 0.16 was found. Whereas low-frequency pulsed electromagnetic fields treated samples showed OD = 1.58 ± 0.24 (P <0.001). However, the percentage of apoptotic cells did not differ between the two groups. CONCLUSIONS: Our data demonstrate that low-frequency pulsed electromagnetic fields emitted by the Somagen™ device influences the in vitro wound healing of patellar tendon fibroblasts and, therefore, possibly increases wound healing potential.

Preventively enteral application of immunoglobulin enriched colostrums milk can modulate postoperative inflammatory response.

Several studies demonstrated acute inflammatory response following traumatic injury. Inflammatory response during surgical interventions was verified by a significant increase of endotoxin plasma levels and a decrease of the endotoxin neutralizing capacity (ENC). However, the incidence of elevated endotoxin levels was significantly higher (89%) than detected bacterial translocation (35%). Thus parts or products of Gram-negative bacteria seem to translocate more easily into the blood circulation than whole bacteria. Along with the bacterial translocation, the inflammatory response correlated directly with the severity of the surgical intervention. In comparison after major and minor surgery Interleukin-6 (IL-6) and C-reactive protein (CRP) was also significantly different. Similar effects in mediator release were shown during endovascular stent graft placement and open surgery in infrarenal aortic aneurysm. Open surgery demonstrated a significant stronger endotoxin translocation and a decrease of ENC. Strategies to prevent translocation seem to be sensible. Colostrum is the first milk produced by the mammary glands within the first days after birth. It contains a complex system of immune factors and has a long history of use in traditional medicine. Placebo-controlled studies verified that prophylactic oral application of immunoglobulin-enriched colostrum milk preparation diminishes perioperative endotoxemia, prevents reduction of ENC and reduces postoperative CRP-levels, suggesting a stabilization of the gut barrier. This effect may be caused by immunoglobulin transportation by the neonatal receptor FcRn of the mucosal epithelium.In conclusion, there is an association of perioperative endotoxemia and the subsequent increase in mediators of the acute phase reaction in surgical patients. A prophylactic oral application of colostrum milk is likely to stabilize the gut barrier i.e. reduces the influx of lipopolysaccharides arising from Gram-negative bacterial pathogens and inhibits enterogenic endotoxemia. This appears to be a major mechanism underlying the therapeutic effect in patients at risk for Gram-negative septic shock.

Light therapy by blue LED improves wound healing in an excision model in rats.

BACKGROUND: Low level light therapy (LLLT) is an attractive alternative to enhance wound healing. So far most studies are performed with red or infrared irradiation. However, we recently showed that blue light (470 nm) can significantly influence biological systems, improving perfusion by release of nitric oxide from nitrosyl complexes with haemoglobin in a skin flap model in rats. Here, we compared the effects of blue and red low level light by light-emitting diodes (LEDs) on in vivo wound healing in an excision wound model in rats. METHODS: Circular excision wounds were surgically created on the dorsum of each rat. Excisions on either the left or right side were illuminated post-OP and on five consecutive days for 10 min by LED at 470 nm or 630 nm with an intensity of 50 mW/cm(2),while protecting the contralateral side from exposure. In the control group, neither side was illuminated. On day 7 post-OP, we analysed planimetric and histological parameters, as well as expression of keratin-1, keratin-10 and keratin-17 on mRNA level. RESULTS: Illumination substantially influenced wound healing. Blue light significantly decreased wound size on day 7, which correlated with enhanced epithelialisation. Light also affected mRNA expression. Both wavelengths decreased keratin-1 mRNA on day 7 post-OP, while keratin-10 mRNA level was elevated in both light treated group compared to control. Keratin-17 mRNA was also elevated in the red light group, but was unchanged in the blue light group. CONCLUSION: In contrast to previous studies, we showed that also blue light significantly influences wound healing. Furthermore, our data suggest that light therapy can play an important role in normotrophic wound healing by affecting keratin expression. Illumination would provide an easily applicable, safe and cost-effective treatment of surface wounds.

Activity of lymphocyte subpopulations in polymicrobial sepsis and DHEA treatment in IL-6 knockout mice.

INTRODUCTION: Sepsis with subsequent multiorgan dysfunction remains the leading cause of mortality in trauma patients. A gender dimorphism in the host response after trauma and sepsis has been revealed. Dehydroepiandrosterone (DHEA), one of the most abundant adrenal sexual steroid hormones, seems to have a protective immunological effect in sepsis. Knowledge of the pathway is sparse; however, a cellular modulation mediated by interleukin-6 (IL-6) has been proposed. MATERIALS AND METHODS: The effect of DHEA on survival, clinical parameters and the cellular immune system (T lymphocytes and NK cells) was examined in a model of polymicrobial sepsis induced by cecal ligation and puncture. For clarification of the role of IL-6 in the protective effect of DHEA, we used IL-6 knockout mice (IL-6(-/-)). As controls, experiments were performed on wild-type mice (WT). RESULTS: The administration of DHEA in IL-6(-/-) mice did not affect mortality, as it was not significantly different from WT mice without DHEA application. The cellular immune response was influenced, as seen by a significant reduction in the percentage of CD8+ and NK cells in WT animals. CONCLUSIONS: Mortality rates in IL-6(-/-) mouse strains were not lowered by DHEA; therefore, a limited effect of IL-6 on this pathway has to be proposed. NK cells may be one of the effector cells of the protective mechanisms of DHEA, whilst the role of CD8+ lymphocytes remains unclear. Consequently, DHEA might be presented as a possible adjuvant therapy after septic insult for modulation of the dysregulated immune system.

A comparison of a cyanoacrylate [corrected] glue (Glubran) vs. fibrin sealant (Tisseel) in experimental models of partial pulmonary resection and lung incision [corrected] in rabbits.

INTRODUCTION: Bronchopleural fistulas (BPF) and air leaks (AL) present major complications after pulmonary resection. Various tissue sealants have been proposed for their prevention, e.g., fibrin sealant (FS) and cyanoacrylate glues (CA). Contrary to the safety record of FS, substantial side effects such as foreign body reaction and impaired tissue integration have been reported for CA. This study compares the sealing efficacy and biocompatibility as well as side effects of FS and CA in experimental partial pulmonary resection and lung incision in rabbits. METHODS: 26 New Zealand white rabbits (3 kg) were randomized to one of the three groups: partial pulmonary resection (A, acute model; n = 7 FS/ 7CA), lung incision [2 (B; n = 3 FS/ 3 CA)], and 14-day observation period (C; n = 3 FS/ 3 CA). In all groups (A, B, and C), FS was considered as control and CA as treatment. Surgery was carried out in general anaesthesia and mechanical ventilation. For partial lung resection a median thoracotomy was performed and the apex of the left median lobe was resected and the parenchymal surface covered with 0.09 ml of FS and CA. The thoracic cavity was filled with ringer solution after 5 minutes. The inspiratory minute volume (IMV) was increased by 0.02 l after every 4th inspiration. In groups B and C, a left lateral thoracotomy was performed in the 4th intercostal space and the left median lobe was incised with a scalpel. The incision was covered with 0.5 ml of FS or CA. At autopsy (B and C) the operation site was assessed macroscopically. Histology was performed in all animals. RESULTS: In terms of sealing purposes, FS and CA yielded comparable results in all groups. CA elicited a substantial increase of tissue temperature in the acute phase immediately after application (A). After 14 days CA residues were found, whereas FS was completely degraded. Histology showed a pronounced inflammatory response to CA but not to FS. We conclude that although the effect of airtight sealing was equally satisfying, our results emphasize that FS is preferable to CA for the prevention of BPF and AL due to superior biocompatibility and degradability. Longterm effects of CA residues on pulmonary tissue require further experimental testing.

Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z RP platform.

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three-dimensional functional tissue-like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three-dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stem cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram. Furthermore, hydroxyapatite coated Sponceram was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP-expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram compared to hydroxyapatite coated Sponceram. With this study, we present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components.

The importance of systemic cytokines in the pathogenesis of polymicrobial sepsis and dehydroepiandrosterone treatment in a rodent model.

The pathogenesis of sepsis is still undetermined to a large extent. It is an established fact that female gender is associated with a lower mortality and that sex steroid hormones influence the immunologic response. Dehydroepiandrosterone (DHEA) seems to have a protective immunologic effect in sepsis. It is still unknown in which way DHEA influences the pathogenesis of sepsis. Therefore, the effect of DHEA application on cytokine concentrations in tumor necrosis factor (TNF) receptor (TNF-RI(-/-)) and interleukin-6 (IL-6(-/-)) knockout mice was determined. In a model of polymicrobial sepsis induced by coecal ligation and puncture (CLP), the effect of DHEA on survival and cytokine concentrations was examined. For clarification of the role of TNF-RI, CLP was performed in TNF-RI knockout mice (TNF-RI(-/-)). In addition, IL-6 knockout mice (IL-6(-/-)) were used to clarify the role of IL-6. Furthermore, experiments were performed in mice that were not genetically modified (wild type, WT). The protective effect of DHEA could be confirmed in this CLP model. DHEA application was associated with a reduction in mortality in WT animals. Moreover, DHEA-treated animals demonstrated a reduction in systemic inflammatory effects, as determined by proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and the antiinflammatory cytokine IL-10. In this work, it was shown that the TNF-RI is essential for survival after CLP. DHEA application was associated with a reduction of mortality of 100% in TNF-RI(-/-) mice after CLP to 50%. This result engages, that the effect of DHEA is TNF-RI independent. However, the application of DHEA had no influence on the mortality in IL-6-/- mice. It can be concluded that the protective effect of DHEA in polymicrobial sepsis is mediated IL-6 dependently. DHEA reduces the systemic inflammation, measurable via the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and the antiinflammatory cytokine IL-10. IL-6 might be involved in the DHEA-mediated reduction of postseptic complications. In contrast, DHEA seems to be TNF-RI independent. Consequently, DHEA might be useful as an adjunct therapy for the immune modulation in sepsis.

Dehydroepiandrosterone (DHEA) modulates the activity and the expression of lymphocyte subpopulations induced by cecal ligation and puncture.

Dehydroepiandrosterone (DHEA) exerts a variety of positive effects on the immunologic alterations after trauma and sepsis. We therefore measured the therapeutic efficacy of DHEA after cecal ligation and puncture (CLP) on the expression of lymphocyte subpopulations and on the delayed type hypersensitivity (DTH) reaction. Male NMRI-mice were randomly assigned to four different treatment groups. Treatment consisted of DHEA or saline (S) administration after CLP or laparotomy only. Flow cytometry was performed (CD4+, CD8+, and CD56 lymphocytes) after 96 hours. DTH-reaction, activity and mortality rate were documented. The CLP-induced reduction in activity and survival (mortality: 34/40) was significantly (p < 0.03) less sustained in CLP-DHEA (mortality: 22/40). The DTH-ratio (before vs. after secondary challenge) was significantly lowered in CLP-S (1.01 +/- 0.15) compared to CLP-DHEA (1.35 +/- 0.1) after 48 hours (p < 0.01). CLP-DHEA (22.2 +/- 7.9%) was associated with a statistically significant less sustained increase of CD56+ cells (p < 0.01) compared with CLP-S (49.0 +/- 6.9%). DHEA-treatment after CLP was associated with less reduction in the CD8+ T-lymphocyte subsets (p < 0.01 vs. all other groups). DHEA treatment after CLP was associated with fewer alterations in the changes of CD8+ and CD56, cells, and the DTH reaction compared with animals submitted to CLP without any treatment. This difference was associated with improved outcome (reactivity, mortality). These results suggest a modulation at specific immune reactions by DHEA treatment.