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Laschke M.W.,Saarland University | Laschke M.W.,Collaborative Research Partner Large Bone Defect Healing Program of AO Foundation | Schank T.E.,Saarland University | Schank T.E.,Collaborative Research Partner Large Bone Defect Healing Program of AO Foundation | And 10 more authors.
Acta Biomaterialia | Year: 2014

Undifferentiated adipose-derived mesenchymal stem cell (adMSC) spheroids are attractive vascularization units for tissue engineering. Their osteogenic differentiation further offers the possibility of directed generation of bone constructs. The aim of this study was to analyze how this differentiation affects their in vivo vascularization capacity. Green fluorescent protein (GFP)-positive adMSCs were isolated from C57BL/6-TgN(ACTB-EGFP)1Osb/J mice for the generation of undifferentiated and differentiated spheroids using the liquid overlay technique. Subsequently, polyurethane scaffolds were seeded with these spheroids and successful osteogenic differentiation was proven by von Kossa staining and high-resolution microtomography. The scaffolds were then implanted into dorsal skinfold chambers of C57BL/6 wild-type mice to analyze their vascularization and incorporation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with differentiated spheroids exhibited a markedly impaired vascularization. Immunohistochemical analyses revealed that this was caused by the lost ability of differentiated spheroids to form GFP-positive microvascular networks inside the scaffolds. This was associated with a reduced tissue incorporation of the implants. Moreover, they no longer exhibited a mineralized matrix after the 14 day implantation period, indicating the dedifferentiation of the spheroids under the given in vivo conditions. These findings indicate that osteogenic differentiation of adMSC spheroids markedly impairs their vascularization capacity. Hence, it may be reasonable to combine adMSC spheroids of varying differentiation stages in scaffolds for bone tissue engineering to promote both vascularization and bone formation. © 2014 Acta Materialia Inc.


Laschke M.W.,Saarland University | Laschke M.W.,Collaborative Research Partner Large Bone Defect Healing Program of AO Foundation | Kleer S.,Saarland University | Kleer S.,Collaborative Research Partner Large Bone Defect Healing Program of AO Foundation | And 6 more authors.
European Cells and Materials | Year: 2015

Adipose tissue-derived microvascular fragments represent promising vascularisation units for implanted tissue constructs. However, their reassembly into functional microvascular networks takes several days, during which the cells inside the implants are exposed to hypoxia. In the present study, we analysed whether this critical phase may be overcome by pre-cultivation of fragment-seeded scaffolds prior to their implantation. Green fluorescent protein (GFP)-positive microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN (ACTB-EGFP) 1Osb/J mice. Nano-size hydroxyapatite particles/poly (ester-urethane) scaffolds were seeded with these fragments and cultivated for 28 days. Subsequently, these scaffolds or control scaffolds, which were freshly seeded with GFP-positive microvascular fragments, were implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study their vascularisation and incorporation by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Pre-cultivation of microvascular fragments resulted in the loss of their native vessel morphology. Accordingly, pre-cultivated scaffolds contained a network of individual CD31/GFP-positive endothelial cells with filigrane cell protuberances. After implantation into the dorsal skinfold chamber, these scaffolds exhibited an impaired vascularisation, as indicated by a significantly reduced functional microvessel density and lower fraction of GFP-positive microvessels in their centre when compared to freshly seeded control implants. This was associated with a deteriorated incorporation into the surrounding host tissue. These findings indicate that freshly isolated, non-cultivated microvascular fragments should be preferred as vascularisation units. This would also facilitate their use in clinical practice during intra-operative one-step procedures. © 2015, AO Research Institute. All rights reserved.


Laschke M.W.,Saarland University | Laschke M.W.,Collaborative Research Partner Large Bone Defect Healing Program of AO Foundation | Grasser C.,Saarland University | Grasser C.,Collaborative Research Partner Large Bone Defect Healing Program of AO Foundation | And 8 more authors.
European Cells and Materials | Year: 2014

Adipose tissue-derived microvascular fragments are promising vascularisation units for applications in the field of tissue engineering. Elderly patients are the major future target population of such applications due to an increasing human life expectancy. Therefore, we herein investigated the effect of aging on the fragments’ vascularisation capacity. Microvascular fragments were isolated from epididymal fat pads of adult (8 months) and aged (16 months) C57BL/6 donor mice. These fragments were seeded onto porous polyurethane scaffolds, which were implanted into dorsal skinfold chambers to study their vascularisation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with fragments from aged donors exhibited a significantly lower functional microvessel density and intravascular blood flow velocity. This was associated with an impaired vessel maturation, as indicated by vessel wall irregularities, constantly elevated diameters and a lower fraction of CD31/α-smooth muscle actin double positive microvessels in the implants’ border and centre zones. Additional in vitro analyses revealed that microvascular fragments from adult and aged donors do not differ in their stem cell content as well as in their release of angiogenic growth factors, survival and proliferative activity under hypoxic conditions. However, fragments from aged donors exhibit a significantly lower number of matrix metalloproteinase -9-positive perivascular cells. Taken together, these findings demonstrate that aging is a crucial determinant for the vascularisation capacity of isolated microvascular fragments. © 2014 AO Research Institute. All rights reserved.

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