Is NO the Answer? The Nitric Oxide Pathway Can Support Bone Morphogenetic Protein 2 Mediated Signaling

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large amounts of BMP2 applied. Therefore, reduction of BMP2 amounts while maintaining efficacy is of clinical importance. As nitric oxide (NO) signaling plays a role in bone fracture healing and an association with the BMP2 pathway has been indicated, this study aimed to investigate the relationship of BMP2 and NO pathways and whether NO can enhance BMP2-induced signaling and osteogenic abilities in vitro. To achieve this, the stable BMP reporter cell line C2C12BRELuc was used to quantify BMP signaling, and alkaline phosphatase (ALP) activity and gene expression were used to quantify osteogenic potency. C2C12BRELuc cells were treated with recombinant BMP2 in combination with NO donors and substrate (Deta NONOate, SNAP & L-Arginine), NOS inhibitor (LNAME), soluble guanylyl cyclase (sGC) inhibitor (LY83583) and activator (YC-1), BMP type-I receptor inhibitor (LDN-193189), or protein kinase A (PKA) inhibitor (H89). It was found that the NOS enzyme, direct NO application, and sGC enhanced BMP2 signaling and improved BMP2 induced osteogenic activity. The application of a PKA inhibitor demonstrated that BMP2 signaling is enhanced by the NO pathway via PKA, underlining the capability of BMP2 in activating the NO pathway. Collectively, this study proves the ability of the NO pathway to enhance BMP2 signaling

Time-Dependent Alterations of MMPs, TIMPs and Tendon Structure in Human Achilles Tendons after Acute Rupture

A balance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) is required to maintain tendon homeostasis. Variation in this balance over time might impact on the success of tendon healing. This study aimed to analyze structural changes and the expression profile of MMPs and TIMPs in human Achilles tendons at different time-points after rupture. Biopsies from 37 patients with acute Achilles tendon rupture were taken at surgery and grouped according to time after rupture: early (2–4 days), middle (5–6 days), and late (≥7 days), and intact Achilles tendons served as control. The histological score increased from the early to the late time-point after rupture, indicating the progression towards a more degenerative status. In comparison to intact tendons, qRT-PCR analysis revealed a significantly increased expression of MMP-1, -2, -13, TIMP-1, COL1A1, and COL3A1 in ruptured tendons, whereas TIMP-3 decreased. Comparing the changes over time post rupture, the expression of MMP-9, -13, and COL1A1 significantly increased, whereas MMP-3 and -10 expression decreased. TIMP expression was not significantly altered over time. MMP staining by immunohistochemistry was positive in the ruptured tendons exemplarily analyzed from early and late time-points. The study demonstrates a pivotal contribution of all investigated MMPs and TIMP-1, but a minor role of TIMP-2, -3, and -4, in the early human tendon healing process. View Full-Tex

An investigation of BMP-7 mediated alterations to BMP signalling components in human tenocyte-like cells

The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers

Bone morphogenetic proteins − 7 and − 2 in the treatment of delayed osseous union secondary to bacterial osteitis in a rat model

Background: Bone infections due to trauma and subsequent delayed or impaired fracture healing represent a great challenge in orthopedics and trauma surgery. The prevalence of such bacterial infection-related types of delayed non-union is high in complex fractures, particularly in open fractures with additional extensive soft-tissue damage. The aim of this study was to establish a rat model of delayed osseous union secondary to bacterial osteitis and investigate the impact of rhBMP-7 and rhBMP-2 on fracture healing in the situation of an ongoing infection. Methods: After randomization to four groups 72 Sprague-Dawley rats underwent a transverse fracture of the midshaft tibia stabilized by intramedullary titanium K-wires. Three groups received an intramedullary inoculation with Staphylococcus aureus (103 colony-forming units) before stabilization and the group without bacteria inoculation served as healing control. After 5 weeks, a second surgery was performed with irrigation of the medullary canal and local rhBMP-7 and rhBMP-2 treatment whereas control group and infected control group received sterile saline. After further 5 weeks rats were sacrificed and underwent biomechanical testing to assess the mechanical stability of the fractured bone. Additional micro-CT analysis, histological, and histomorphometric analysis were done to evaluate bone consolidation or delayed union, respectively, and to quantify callus formation and the mineralized area of the callus. Results: Biomechanical testing showed a significantly higher fracture torque in the non-infected control group and the infected rhBMP-7- and rhBMP-2 group compared with the infected control group (p < 0.001). RhBMP-7 and rhBMP-2 groups did not show statistically significant differences (p = 0.57). Histological findings supported improved bone-healing after rhBMP treatment but quantitative micro-CT and histomorphometric results still showed significantly more hypertrophic callus tissue in all three infected groups compared to the non-infected group. Results from a semiquantitative bone-healing-score revealed best bone-healing in the non-infected control group. The expected chronic infection was confirmed in all infected groups. Conclusions: In delayed bone healing secondary to infection rhBMP treatment promotes bone healing with no significant differences in the healing efficacy of rhBMP-2 and rhBMP-7 being noted. Further new therapeutic bone substitutes should be analyzed with the present rat model for delayed osseous union secondary to bacterial osteitis

Untersuchung von Signalwegen in Rattenmodellen mit unterschiedlichen Knochenheilungsstörungen

Trotz verbesserter operativer Versorgung und fundiertem Wissen über die Heilungsabläufe, treten auch heute noch schwerwiegende Komplikationen während der Knochenheilung auf. Daher ist es von außerordentlicher Bedeutung neue Erkenntnisse zur Pathologie der Knochenheilungsstörung zu gewinnen. Eine Möglichkeit ist der Vergleich unterschiedlicher, klinisch relevanter Tiermodelle. Diese Promotion beschäftigt sich mit der Analyse von drei Heilungsmodellen, die eine i. physiologische Knochenheilung (Kontrollgruppe), ii. hypertrophe Pseudarthrose (Hypertrophiegruppe) und iii. atrophe Pseudarthrose (Atrophiegruppe) imitieren. Dabei wurde die Expression osteogener und angiogener Signalwegkomponenten vergleichend mittels mRNA Analyse und Immunhistochemie sowie die Revaskularisierung des Kallusgewebes untersucht. Der Wechsel von einem geschlossenen Frakturansatz (Kontrollgruppe) zu einer offenen Osteotomie (Hypertrophiegruppe), und damit Setzen eines zusätzlichen Weichteiltraumas, führte bereits zu einer verlängerten Heilung mit fehlender knöcherner Überbrückung nach 42 Tagen. qRT-PCR Daten zeigten eine höhere Expression der analysierten osteogenen und angiogenen Faktoren in der Hypertrophiegruppe zu Tag 14. Nach 42 Tagen kam es zu einer signifikanten Reduktion osteogener Faktoren und deren Antagonisten wie Bmp4 und Bambi. Die Inhibierung der Angiogenese durch Fumagillin (Atrophiegruppe) verringerte die Bildung neuer Blutgefäße sowie die Chondrogenese in spaltnahen Bereichen und führte nach 42 Tagen zu einer kompletten Nichtheilungssituation. Angiogene Faktoren zeigten eine Hochregulation über die späteren Heilungszeitpunkte und gleichzeitig waren osteogene Faktoren weniger reguliert zu Tag 14 und 21 im Vergleich zur Kontrollgruppe. Bis heute gibt es keine Studie, die sich mit dem Vergleich mehrerer verzögerter Heilungsmodelle, in Hinblick auf die Unterschiede im osteogenen und angiogenen Signalweg, beschäftigt hat. Besonders aus gesundheitsökonomischer Sicht ist es von außerordentlicher Bedeutung, individuelle Therapieformen zu entwickeln und dabei die Kosten der Therapie so gering wie möglich zu halten. Ein wichtiger Schritt in diese Richtung ist das Verständnis über die Ursachen und sich anschließende Regulationsmechanismen in den unterschiedlichen Knochenheilungsstörungen vor allem auf molekularer Ebene. Das Projekt zur Untersuchung klinisch relevanter Knochenheilungsmodelle am Tier stellt dabei eine enge Verknüpfung von Forschung und Klinik dar

Time-Dependent Alterations of MMPs, TIMPs and Tendon Structure in Human Achilles Tendons after Acute Rupture

A balance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) is required to maintain tendon homeostasis. Variation in this balance over time might impact on the success of tendon healing. This study aimed to analyze structural changes and the expression profile of MMPs and TIMPs in human Achilles tendons at different time-points after rupture. Biopsies from 37 patients with acute Achilles tendon rupture were taken at surgery and grouped according to time after rupture: early (2–4 days), middle (5–6 days), and late (≥7 days), and intact Achilles tendons served as control. The histological score increased from the early to the late time-point after rupture, indicating the progression towards a more degenerative status. In comparison to intact tendons, qRT-PCR analysis revealed a significantly increased expression of MMP-1, -2, -13, TIMP-1, COL1A1, and COL3A1 in ruptured tendons, whereas TIMP-3 decreased. Comparing the changes over time post rupture, the expression of MMP-9, -13, and COL1A1 significantly increased, whereas MMP-3 and -10 expression decreased. TIMP expression was not significantly altered over time. MMP staining by immunohistochemistry was positive in the ruptured tendons exemplarily analyzed from early and late time-points. The study demonstrates a pivotal contribution of all investigated MMPs and TIMP-1, but a minor role of TIMP-2, -3, and -4, in the early human tendon healing process

Investigation of signaling pathways in different clinically relevant rat bone healing mode

Trotz verbesserter operativer Versorgung und fundiertem Wissen über die Heilungsabläufe, treten auch heute noch schwerwiegende Komplikationen während der Knochenheilung auf. Daher ist es von außerordentlicher Bedeutung neue Erkenntnisse zur Pathologie der Knochenheilungsstörung zu gewinnen. Eine Möglichkeit ist der Vergleich unterschiedlicher, klinisch relevanter Tiermodelle. Diese Promotion beschäftigt sich mit der Analyse von drei Heilungsmodellen, die eine i. physiologische Knochenheilung (Kontrollgruppe), ii. hypertrophe Pseudarthrose (Hypertrophiegruppe) und iii. atrophe Pseudarthrose (Atrophiegruppe) imitieren. Dabei wurde die Expression osteogener und angiogener Signalwegkomponenten vergleichend mittels mRNA Analyse und Immunhistochemie sowie die Revaskularisierung des Kallusgewebes untersucht. Der Wechsel von einem geschlossenen Frakturansatz (Kontrollgruppe) zu einer offenen Osteotomie (Hypertrophiegruppe), und damit Setzen eines zusätzlichen Weichteiltraumas, führte bereits zu einer verlängerten Heilung mit fehlender knöcherner Überbrückung nach 42 Tagen. qRT-PCR Daten zeigten eine höhere Expression der analysierten osteogenen und angiogenen Faktoren in der Hypertrophiegruppe zu Tag 14. Nach 42 Tagen kam es zu einer signifikanten Reduktion osteogener Faktoren und deren Antagonisten wie Bmp4 und Bambi. Die Inhibierung der Angiogenese durch Fumagillin (Atrophiegruppe) verringerte die Bildung neuer Blutgefäße sowie die Chondrogenese in spaltnahen Bereichen und führte nach 42 Tagen zu einer kompletten Nichtheilungssituation. Angiogene Faktoren zeigten eine Hochregulation über die späteren Heilungszeitpunkte und gleichzeitig waren osteogene Faktoren weniger reguliert zu Tag 14 und 21 im Vergleich zur Kontrollgruppe. Bis heute gibt es keine Studie, die sich mit dem Vergleich mehrerer verzögerter Heilungsmodelle, in Hinblick auf die Unterschiede im osteogenen und angiogenen Signalweg, beschäftigt hat. Besonders aus gesundheitsökonomischer Sicht ist es von außerordentlicher Bedeutung, individuelle Therapieformen zu entwickeln und dabei die Kosten der Therapie so gering wie möglich zu halten. Ein wichtiger Schritt in diese Richtung ist das Verständnis über die Ursachen und sich anschließende Regulationsmechanismen in den unterschiedlichen Knochenheilungsstörungen vor allem auf molekularer Ebene. Das Projekt zur Untersuchung klinisch relevanter Knochenheilungsmodelle am Tier stellt dabei eine enge Verknüpfung von Forschung und Klinik dar.Severe complications in bone healing, such as delayed healing or non-union progressions, are still present although a great deal of information about the general healing process and treatment strategies are available. Therefore, it is of crucial importance to gain an insight into the pathology of healing failure. One possibility is to compare different clinical relevant animal models. The aim of this study was to evaluate biological characteristics of hypertrophic and atrophic non unions in comparison to a physiological healing outcome. In all three healing models expression levels of relevant pathway components important for osteogenic differentiation and angiogenesis were analyzed using mRNA analysis and immunohistochemistry. Additionally, the revascularization during the entire healing process was investigated ex vivo by utilization of µCT technique in combination with the application of a polymerizing contrast agent. The change from a closed fracture approach (control group) to an open osteotomy (hypertrophy group), and thereby inducing a soft tissue damage, led to a prolonged healing with a lack in bony bridging after 42 days. qRT-PCR data revealed higher expression of osteogenic and angiogenic genes at day 14. After 42 days a significant reduction of osteogenic factors and their antagonists such as Bmp4 and Bambi could be seen. The inhibition of angiogenesis by the application of Fumagillin (atrophy group) not only reduced the number of vessels, but inhibited chondrogenesis in gap regions and led to a complete non-union situation after 42 days. Angiogenic factors were highly upregulated over a longer time whereas osteogenic genes were less regulated compared to the control group. Up to now, no study compared three different clinically relevant bone healing models focusing on both the osteogenic and angiogenic signaling pathway. Understanding the cause and the associated regulation mechanism on molecular level are important steps towards development of individual treatment strategies. This project tightly links science and clinical needs by analyzing clinically relevant bone healing progressions in an animal model

Is NO the Answer? The Nitric Oxide Pathway Can Support Bone Morphogenetic Protein 2 Mediated Signaling

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large amounts of BMP2 applied. Therefore, reduction of BMP2 amounts while maintaining efficacy is of clinical importance. As nitric oxide (NO) signaling plays a role in bone fracture healing and an association with the BMP2 pathway has been indicated, this study aimed to investigate the relationship of BMP2 and NO pathways and whether NO can enhance BMP2-induced signaling and osteogenic abilities in vitro. To achieve this, the stable BMP reporter cell line C2C12BRELuc was used to quantify BMP signaling, and alkaline phosphatase (ALP) activity and gene expression were used to quantify osteogenic potency. C2C12BRELuc cells were treated with recombinant BMP2 in combination with NO donors and substrate (Deta NONOate, SNAP & L-Arginine), NOS inhibitor (LNAME), soluble guanylyl cyclase (sGC) inhibitor (LY83583) and activator (YC-1), BMP type-I receptor inhibitor (LDN-193189), or protein kinase A (PKA) inhibitor (H89). It was found that the NOS enzyme, direct NO application, and sGC enhanced BMP2 signaling and improved BMP2 induced osteogenic activity. The application of a PKA inhibitor demonstrated that BMP2 signaling is enhanced by the NO pathway via PKA, underlining the capability of BMP2 in activating the NO pathway. Collectively, this study proves the ability of the NO pathway to enhance BMP2 signaling