Adipose tissue-derived microvascular fragments: In vitro and in vivo analysis of prevascularization strategies for dermal substitutes

For the treatment of full-thickness skin defects, autologous split-thickness skin grafting is the gold standard. However, the outcome of this approach may be impaired by scarring, wound contraction, or the lack of vascularization within the wound bed. To counteract these problems, bioengineered off-the-shelf matrices, such as Integra® Matrix Wound Dressing (MWD), have been introduced over the past years. This collagen-glycosaminoglycan matrix with a silicone pseudo-epidermis is first implanted into the debrided wound bed. Only after a sufficient vascularization, the silicone layer is removed and the matrix can be covered with a split-thickness skin graft. Noteworthy, the ingrowth of blood vessels into the matrix requires up to 3 weeks. Since wound infection is a threat throughout this time, strategies are needed to improve the vascularization of the matrix. Prevascularization, which aims for the creation of microvascular networks within the matrix prior to its implantation, is such a strategy. Although the seeding of matrices with stem cells showed positive results in the creation of preformed networks, a time-consuming in vitro cultivation is necessary to generate the needed number of cells. Moreover, stem cells need too much time to vascularize the entire matrix. To overcome this problem, the seeding of matrices with adipose tissue-derived microvascular fragments (ad-MVF) has recently been introduced as a promising strategy. Ad-MVF can easily be isolated from adipose tissue and, unlike single cells, already represent fully functional blood vessel segments that rapidly reassemble into new microvascular networks within seeded matrices. However, although experimental studies indicate the benefits of ad-MVF as prevascularization units, many questions have to be clarified to allow a successful transfer of this novel approach into clinical practice, as outlined and analyzed in the present doctoral thesis. For all in vitro and in vivo parts of this thesis, green fluorescent protein (GFP)+ C57BL/6 mice were used as ad-MVF donor animals. The tissue of these animals appears green under blue light excitation, enabling the identification of ad-MVF after their transplantation into GFP- C57BL/6 recipient mice. Non-seeded and ad-MVF- or stromal vascular fraction (SVF)- seeded Integra® matrices were implanted into full-thickness skin defects within mouse dorsal skinfold chambers to analyze their vascularization, incorporation, oxygenation, and epithelialization by means of intravital fluorescence microscopy, photoacoustic imaging, histology, and immunohistochemistry over an observation period of 2 weeks. The aim of a first set of experiments was to clarify whether the volumes of epididymal adipose tissue and/or the numbers of ad-MVF depend on the age of the animal. For this purpose, 7 - 12 months old GFP+ C57BL/6 donor mice were used for harvesting their epididymal fat tissue and subsequent ad-MVF isolations. It was found that a comparable volume of ~ 1.4 mL fat tissue can be harvested from animals ranging between 7 - 12 months. Furthermore, a comparable number of ~ 40.000 ad-MVF/mL adipose tissue could be isolated from these mice. The isolated ad-MVF exhibited an average length of ~ 38 μm and a viability of > 90 %. This indicates that all mice of the analyzed age range can be used for a highly standardized isolation of ad-MVF. In a second set of experiments it was hypothesized that the seeding of ad-MVF on a flowable matrix, which allows a more homogeneous distribution, may result in a faster matrix vascularization and incorporation when compared to MWD. For this purpose, MWD as well as samples of Integra® Flowable Wound Matrix (FWM) were seeded with identical numbers of ad-MVF. Of interest, FWM exhibited a more homogeneous distribution of individual ad- MVF when compared to MWD. However, when implanted into full-thickness skin defects within GFP- C57BL/6 recipient mice, this resulted in an increased distance between individual ad-MVF, leading to a prolonged ad-MVF interconnection time as well as a reduced matrix vascularization and incorporation in vivo. These results indicate that MWD is more suitable for the treatment of rather large wounds, while FWM may be particularly used for the treatment of small wounds and wounds with irregular geometries which are hardly to access. In a third set of experiments it was hypothesized that the seeding density of ad-MVF is a crucial determinant for an adequate vascularization and incorporation of seeded matrices. For this purpose, MWD was seeded with three different densities of ad-MVF. The results indicate that a minimum density of ~ 80,000 ad-MVF/cm2 is required to guarantee a sufficient vascularization and incorporation of implanted MWD in vivo. Under clinical conditions, this may be particularly relevant for the treatment of patients with extensive skin defects and/or limited availability of adipose tissue. Besides the novel approach of ad-MVF prevascularization, the seeding of matrices with vessel-forming single cells of the SVF is a frequently applied strategy. In a fourth set of experiments it was hypothesized that the ad-MVF-based prevascularization of MWD is superior to the conventional SVF-based approach. While both ad-MVF and SVF isolates contained a comparable fraction of endothelial cells, perivascular cells, adipocytes, and stem cells, ad-MVF exhibited a significantly higher viability after their isolation. It was further shown that SVF-seeded MWD exhibited a reduced vascularization and incorporation when compared to ad-MVF-seeded MWD in vivo. These novel findings indicate that ad-MVF are highly potent vascularization units that may be even superior to SVF single cells in future tissue engineering applications. In a fifth set of experiments it was hypothesized that the anti-angiogenic properties of low molecular weight heparins (LMWH), such as enoxaparin (enox), prevent the reassembly of seeded ad-MVF into new microvascular networks. It was further hypothesized that the anticoagulative properties of enox may increase the bleeding risk during network formation within implanted MWD. To test these hypotheses, ad-MVF were isolated from enox- and vehicle-treated GFP+ C57BL/6 donor mice, seeded onto MWD and implanted into enox- and vehicle-treated GFP- recipient animals. Intravital fluorescence microscopic, histological, and immunohistochemical analyses revealed that enox does neither inhibit the reassembly of ad- MVF nor promotes implant-induced hemorrhage formation. These novel findings demonstrate that the seeding of MWD matrices with ad-MVF as prevascularization units may also be applied during thromboprophylactic therapy without any concern.Obwohl die Deckung dermaler Wunden mittels autologer Spalthauttransplantate zur klinischen Standardbehandlung zählt, kann der Erfolg dieser Methode durch Komplikationen wie Narbenbildung, Wundkontraktionen oder eine ausbleibende Vaskularisierung des Wundbetts gefährdet werden. Um diese Probleme zu vermeiden, wurden in den letzten Jahren biosynthetische Matrices, wie beispielsweise Integra® Matrix Wound Dressing (MWD), entwickelt. MWD ist eine Kollagen-Glykosaminoglykan-Matrix, welche mit einer Pseudo-Epidermis aus Silikon bedeckt ist, um die Austrocknung der Wunde zu verhindern. Erst nach ausreichender Vaskularisierung der Matrix kann die Silikonschicht abgenommen werden und eine Deckung mit Spalthaut erfolgen. Da die Vaskularisierung bis zu 3 Wochen dauern kann und in diesem Zeitraum ein erhöhtes Infektionsrisiko besteht, müssen unbedingt neue Strategien zur Beschleunigung der Vaskularisierung solcher Matrices entwickelt werden. Ein solcher Ansatz ist die Prävaskularisierung, eine Methode, bei der mikrovaskuläre Netzwerke in einer Matrix vor deren Implantation generiert werden. Auch wenn die Besiedlung von Matrices mit Stammzellen bereits positive Ergebnisse bei der Herstellung prävaskularisierter Netzwerke gezeigt hat, sind dabei zeitaufwändige in vitro Kultivierungen unabdingbar, um eine ausreichende Zellzahl zu erhalten. Darüber hinaus benötigen Stammzellen zu viel Zeit, bis sie sich in der gesamten Matrix zu neuen Blutgefäßen entwickelt haben. Um diese Probleme zu vermeiden, wurde kürzlich die Prävaskularisierung von Matrices mit mikrovaskulären Fragmenten (ad-MVF) aus Fettgewebe als neue Strategie beschrieben. Ad-MVF können in großen Mengen durch enzymatische Digestion von Fettgewebe gewonnen werden und stellen, im Gegensatz zu Einzelzellen, bereits intakte Gefäßsegmente dar, welche sich innerhalb einer Matrix nur noch zusammenlagern müssen, um vollständige Netzwerke auszubilden. Auch wenn experimentelle Studien den Einsatz von ad-MVF als Prävaskularisierungseinheiten bereits als vielversprechend beschreiben, müssen noch wesentliche Fragen beantwortet werden, um diesen Ansatz erfolgreich in die Klinik einzuführen. Für alle in vitro und in vivo Versuche der vorliegenden Arbeit wurden green fluorescent protein (GFP)+ C57BL/6 Spendermäuse zur Fettgewinnung und Isolation von ad-MVF verwendet. Das Gewebe dieser Tiere erscheint unter Anregung mit blauem Licht grün und ermöglicht so die Identifikation von ad-MVF nach Implantation in GFP- Empfängertiere. Unbesiedelte und ad-MVF- oder stromal vascular fraction (SVF)-besiedelte Integra® Matrices wurden in Vollhautdefekte innerhalb von Rückenhautkammern implantiert und anschließend deren Vaskularisierung, Inkorporation, Oxygenierung und Epithelialisierung mittels intravitaler Fluoreszenzmikroskopie, photoakustischer Bildgebung, Histologie und Immunhistochemie über einen Zeitraum von 2 Wochen analysiert. In einem ersten experimentellen Teil wurde die Hypothese geprüft, ob die Menge an epididymalem Fettgewebe oder die Anzahl isolierbarer ad-MVF abhängig vom Alter des Spendertieres sind. Zu diesem Zweck wurden 7 - 12 Monate alte GFP+ C57BL/6 Mäuse verwendet, um deren epididymales Fett zu entnehmen und ad-MVF zu isolieren. Dabei konnte gezeigt werden, dass aus allen Spendermäusen ein vergleichbares Volumen von ~ 1,4 ml Fettgewebe gewonnen werden kann. Zusätzlich war es bei einer Viabilität von > 90 % möglich, aus allen 7 - 12 Monate alten Tieren ~ 40,000 ad-MVF/ml Fettgewebe zu isolieren, wobei diese eine mittlere Länge von ~ 38 μm aufwiesen. Diese Ergebnisse zeigen, dass aus 7 - 12 Monate alten Spendermäusen eine standardisierte Isolation von ad-MVF möglich ist. In einem zweiten experimentellen Teil wurde die Hypothese geprüft, ob eine homogenere Verteilung von ad-MVF in einer fließfähigen Matrix zu einer verbesserten in vivo Vaskularisierung führt. Hierzu wurden sowohl MWD als auch Einzelproben der Integra® Flowable Wound Matrix (FWM) mit einer identischen Anzahl von ad-MVF besiedelt. Auf diese Weise konnte gezeigt werden, dass FWM eine homogenere Verteilung von ad-MVF ermöglicht. Dies ging allerdings mit einer erhöhten Distanz einzelner ad-MVF zueinander einher und führte zu einer verlangsamten Netzwerkbildung. Entsprechend war auch die Vaskularisierung und Inkorporation der Matrices verzögert. Dieses Ergebnis zeigt, dass MWD zur Behandlung größerer Wunden eingesetzt werden sollte, während ad-MVFbesiedelte FWM eher in kleinen, irregulär geformten Wunden Anwendung finden könnte. In einem dritten experimentellen Teil wurde die Hypothese geprüft, ob eine zu geringe Dichte von ad-MVF bewirkt, dass eine ausreichende Vaskularisierung implantierter Matrices nicht mehr erfolgen kann. Hierzu wurden MWD mit 3 verschiedenen Dichten von ad-MVF besiedelt. Es konnte gezeigt werden, dass eine Mindestdichte von ~ 80,000 ad-MVF/cm2 MWD notwendig ist, um eine ausreichende Vaskularisierung und Inkorporation der Matrices zu erreichen. Dies ist im Speziellen für klinische Anwendungen wichtig, bei denen am Patienten große Defekte gedeckt werden müssen und/oder ein eingeschränktes Volumen an Fettgewebe für die Isolierung von ad-MVF vorhanden ist. Neben dem Besiedeln von Matrices mit ad-MVF beschreibt der Einsatz der SVF aus Fettgewebe eine klinisch bereits angewandte Methode der Prävaskularisierung. In einem vierten experimentellen Teil wurde die Hypothese geprüft, ob eine Prävaskularisierung mit ad-MVF dem konventionellen SVF-basierten Ansatz überlegen ist. Obwohl ad-MVF- und SVF-Isolate vergleichbare Anteile an Endothelzellen, perivaskulären Zellen, Adipozyten und Stammzellen aufwiesen, war die Viabilität von frisch isolierten ad-MVF höher. Weiterhin konnte gezeigt werden, dass SVF-besiedelte Matrices eine schlechtere Vaskularisierung aufweisen. Diese Ergebnisse zeigen, dass auf Grund ihres hohen Vaskularisierungspotentials ad-MVF gegenüber SVF-Einzelzellen für zukünftige Tissue Engineering-Ansätze deutlich besser geeignet sein könnten. In einem fünften experimentellen Teil wurde die Hypothese geprüft, ob die anti-angiogene Wirkung von niedermolekularen Heparinen (LMWH), wie Enoxaparin (enox), die Netzwerkbildung von ad-MVF verlangsamt. Weiterhin wurde hypothetisiert, dass die antikoagulativen Eigenschaften von enox zu vermehrten Einblutungen während der Netzwerkausbildung innerhalb ad-MVF-besiedelter Matrices führen. Hierzu wurden ad-MVF von GFP+ C57BL/6 Mäusen isoliert, welche mit enox oder Vehikel behandelt wurden. Anschließend wurde MWD mit diesen ad-MVF besiedelt und passgenau in Vollhautdefekte innerhalb des Rückenhautkammermodells implantiert. Intravitalmikroskopische, histologische, und immunhistochemische Analysen konnten zeigen, dass enox weder die Netzwerkbildung von ad-MVF verschlechtert, noch Einblutungen innerhalb der Matrices begünstigt. Diese Ergebnisse belegen, dass eine Besiedelung von MWD mit ad-MVF als Prävaskularisierungseinheiten bei gleichzeitig angewandter Thromboseprophylaxe problemlos möglich ist

The Marine-Derived Triterpenoid Frondoside A Inhibits Thrombus Formation

Background: The marine-derived triterpenoid frondoside A inhibits the phosphatidylinositol-3-kinase (PI3K) pathway in cancer cells. Because this pathway is also crucially involved in platelet activation, we studied the effect of frondoside A on thrombus formation. Methods: Frondoside A effects on platelet viability, surface adhesion molecule expression, and intracellular signaling were analyzed by flow cytometry and Western blot. The effect of frondoside A was analyzed by photochemically induced thrombus formation in the mouse dorsal skinfold chamber model and by tail vein bleeding. Results: Concentrations of up to 15 µM frondoside A did not affect the viability of platelets, but reduced their surface expression of P-selectin (CD62P) and the activation of glycoprotein (GP)IIb/IIIa after agonist stimulation. Additional mechanistic analyses revealed that this was mediated by downregulation of PI3K-dependent Akt and extracellular-stimuli-responsive kinase (ERK) phosphorylation. Frondoside A significantly prolonged the complete vessel occlusion time in the mouse dorsal skinfold chamber model of photochemically induced thrombus formation and also the tail vein bleeding time when compared to vehicle-treated controls. Conclusion: Our findings demonstrated that frondoside A inhibits agonist-induced CD62P expression and activation of GPIIb/IIIa. Moreover, frondoside A suppresses thrombus formation. Therefore, this marine-derived triterpenoid may serve as a lead compound for the development of novel antithrombotic drugs

Macrophages promote network formation and maturation of transplanted adipose tissue-derived microvascular fragments

Adipose tissue-derived microvascular fragments rapidly reassemble into microvascular networks within implanted scaffolds. Herein, we analyzed the contribution of macrophages to this process. C57BL/6 mice received clodronate (clo)-containing liposomes for macrophage depletion, whereas animals treated with phosphate-buffered-saline-containing liposomes served as controls. Microvascular fragments were isolated from clo- and phosphate-buffered-saline-treated donor mice and seeded onto collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of clo- and phosphate-buffered-saline-treated recipient mice. The implants' vascularization and incorporation were analyzed by stereomicroscopy, intravital fluorescence microscopy, histology, and immunohistochemistry. Compared to controls, matrices within clo-treated animals exhibited a significantly reduced functional microvessel density. Moreover, they contained a lower fraction of microvessels with an α-smooth muscle actin (SMA)+ cell layer, indicating impaired vessel maturation. This was associated with a deteriorated implant incorporation. These findings demonstrate that macrophages not only promote the reassembly of microvascular fragments into microvascular networks, but also improve their maturation during this process

The Marine-Derived Kinase Inhibitor Fascaplysin Exerts Anti-Thrombotic Activity

Background: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis. Methods: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time. Results: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time. Conclusion: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound

Vascularization of Microvascular Fragment Isolates from Visceral and Subcutaneous Adipose Tissue of Mice

Background: Adipose tissue-derived microvascular fragments (MVF) represent effective vascularization units for tissue engineering. Most experimental studies in rodents exclusively use epididymal adipose tissue as a visceral fat source for MVF isolation. However, in future clinical practice, MVF may be rather isolated from liposuctioned subcutaneous fat tissue of patients. Therefore, we herein compared the vascularization characteristics of MVF isolates from visceral and subcutaneous fat tissue of murine origin. Methods: MVF isolates were generated from visceral and subcutaneous fat tissue of donor mice using two different enzymatic procedures. For in vivo analyses, the MVF isolates were seeded onto collagen-glycosaminoglycan scaffolds and implanted into full-thickness skin defects within dorsal skinfold chambers of recipient mice. Results: By means of the two isolation procedures, we isolated a higher number of MVF from visceral fat tissue when compared to subcutaneous fat tissue, while their length distribution, viability and cellular composition were comparable in both groups. Intravital fluorescence microscopy as well as histological and immunohistochemical analyses revealed a significantly reduced vascularization of implanted scaffolds seeded with subcutaneous MVF isolates when compared to implants seeded with visceral MVF isolates. Light and scanning electron microscopy showed that this was due to high amounts of undigested connective tissue within the subcutaneous MVF isolates, which clogged the scaffold pores and prevented the interconnection of individual MVF into new microvascular networks. Conclusion: These findings indicate the need for improved protocols to generate connective tissue-free MVF isolates from subcutaneous fat tissue for future translational studies

Pantoprazole impairs fracture healing in aged mice

Proton pump inhibitors (PPIs) belong to the most common medication in geriatric medicine. They are known to reduce osteoclast activity and to delay fracture healing in young adult mice. Because differentiation and proliferation in fracture healing as well as pharmacologic actions of drugs markedly differ in the elderly compared to the young, we herein studied the effect of the PPI pantoprazole on bone healing in aged mice using a murine fracture model. Bone healing was analyzed by biomechanical, histomorphometric, radiological and protein biochemical analyses. The biomechanical analysis revealed a significantly reduced bending stiffness in pantoprazole-treated animals when compared to controls. This was associated with a decreased amount of bone tissue within the callus, a reduced trabecular thickness and a higher amount of fibrous tissue. Furthermore, the number of osteoclasts in pantoprazole-treated animals was significantly increased at 2 weeks and decreased at 5 weeks after fracture, indicating an acceleration of bone turnover. Western blot analysis showed a lower expression of the bone morphogenetic protein-4 (BMP-4), whereas the expression of the pro-angiogenic parameters was higher when compared to controls. Thus, pantoprazole impairs fracture healing in aged mice by affecting angiogenic and osteogenic growth factor expression, osteoclast activity and bone formation

Improvement of islet transplantation by the fusion of islet cells with functional blood vessels

Pancreatic islet transplantation still represents a promising therapeutic strategy for curative treatment of type 1 diabetes mellitus. However, a limited number of organ donors and insufficient vascularization with islet engraftment failure restrict the successful transfer of this approach into clinical practice. To overcome these problems, we herein introduce a novel strategy for the generation of prevascularized islet organoids by the fusion of pancreatic islet cells with functional native microvessels. These insulin-secreting organoids exhibit a significantly higher angiogenic activity compared to freshly isolated islets, cultured islets, and non-prevascularized islet organoids. This is caused by paracrine signaling between the β-cells and the microvessels, mediated by insulin binding to its corresponding receptor on endothelial cells. In vivo, the prevascularized islet organoids are rapidly blood-perfused after transplantation by the interconnection of their autochthonous microvasculature with surrounding blood vessels. As a consequence, a lower number of islet grafts are required to restore normoglycemia in diabetic mice. Thus, prevascularized islet organoids may be used to improve the success rates of clinical islet transplantation

Conditional Expression of Wnt4 during Chondrogenesis Leads to Dwarfism in Mice

Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26(floxneoWnt4). Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26(floxneoWnt4); Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26(floxneoWnt4); Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF). These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype

Engineering microparticles based on solidified stem cell secretome with an augmented pro-angiogenic factor portfolio for therapeutic angiogenesis

Tissue (re)vascularization strategies face various challenges, as therapeutic cells do not survive long enough in situ, while the administration of pro-angiogenic factors is hampered by fast clearance and insufficient ability to emulate complex spatiotemporal signaling. Here, we propose to address these limitations by engineering a functional biomaterial capable of capturing and concentrating the pro-angiogenic activities of mesenchymal stem cells (MSCs). In particular, dextran sulfate, a high molecular weight sulfated glucose polymer, supplemented to MSC cultures, interacts with MSC-derived extracellular matrix (ECM) components and facilitates their co-assembly and accumulation in the pericellular space. Upon decellularization, the resulting dextran sulfate-ECM hybrid material can be processed into MIcroparticles of SOlidified Secretome (MIPSOS). The insoluble format of MIPSOS protects protein components from degradation, while facilitating their sustained release. Proteomic analysis demonstrates that MIPSOS are highly enriched in pro-angiogenic factors, resulting in an enhanced pro-angiogenic bioactivity when compared to naïve MSC-derived ECM (cECM). Consequently, intravital microscopy of full-thickness skin wounds treated with MIPSOS demonstrates accelerated revascularization and healing, far superior to the therapeutic potential of cECM. Hence, the microparticle-based solidified stem cell secretome provides a promising platform to address major limitations of current therapeutic angiogenesis approaches

Transcriptional Analysis of Fracture Healing and the Induction of Embryonic Stem Cell–Related Genes

Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ∼350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs