Amlodipine accelerates bone healing in a stable closed femoral fracture model in mice.

Calcium channel blockers (CCBs), which are widely used in the treatment of hypertension, have been shown to influence bone metabolism. However, there is little information on whether CCBs also influence the process of fracture healing. Therefore, the effect of the CCB amlodipine on bone healing was studied in a stable closed fracture model in mice using intramedullary screw fixation. Bone healing was investigated by radiology, biomechanics, histomorphometry and Western blot analysis 2 and 5 weeks after fracture healing. Animals were treated daily (post operatively) per os using a gavage with amlodipine low dose (1 mg/ kg body weight, n = 20), amlodipine high dose (3 mg/kg body weight, n = 20) or vehicle (NaCl) (control, n = 20) serving as a negative control. At 2 and 5 weeks, histomorphometric analysis revealed a significantly larger amount of bone tissue within the callus of amlodipine low-dose- and high-dose-treated animals when compared to controls. This was associated with a smaller amount of cartilaginous and fibrous tissue, indicating an acceleration of fracture healing. Biomechanics showed a slightly, but not significantly, higher bending stiffness in amlodipine low-dose- and high-dose-treated animals. Western blot analysis revealed a significantly increased expression of bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF). Moreover, the analysis showed a 5-fold higher expression of osteoprotegerin (OPG) and a 10-fold elevated expression of the receptor activator of NF-κB ligand (RANKL), indicating an increased bone turnover. These findings demonstrated that amlodipine accelerated fracture healing by stimulating bone formation, callus remodelling and osteoclast activity.

Erythropoietin promotes network formation of transplanted adipose tissue-derived microvascular fragments.

The seeding of tissue constructs with adipose tissue-derived microvascular fragments (ad-MVF) is an emerging pre-vascularisation strategy. Ad-MVF rapidly reassemble into new microvascular networks after in vivo implantation. Herein it was analysed whether this process was improved by erythropoietin (EPO). Ad-MVF were isolated from green fluorescent protein (GFP)+ as well as wild-type C57BL/6 mice and cultivated for 24 h in medium supplemented with EPO (20 IU/mL) or vehicle. Freshly isolated, non-cultivated ad-MVF served as controls. Protein expression, cell viability and proliferation of ad-MVF were assessed by proteome profiler array and fluorescence microscopy. GFP+ ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of C57BL/6 mice, to analyse their vascularisation over 14 d by intravital fluorescence microscopy, histology and immunohistochemistry. Cultivation up-regulated the expression of pro- and anti-angiogenic factors within both vehicle- and EPO-treated ad-MVF when compared with non-cultivated controls. Moreover, EPO treatment suppressed cultivation-associated apoptosis and significantly increased the number of proliferating endothelial cells in ad-MVF when compared with vehicle-treated and non-cultivated ad-MVF. Accordingly, implanted matrices seeded with EPO-treated ad-MVF exhibited an improved vascularisation, as indicated by a significantly higher functional microvessel density. The matrices of the three groups contained a comparably large fraction of GFP+ microvessels originating from the ad-MVF, whereas the tissue surrounding the matrices seeded with EPO-treated ad-MVF exhibited a significantly increased microvessel density when compared with the other two groups. These findings indicated that EPO represents a promising cytokine to further boost the excellent vascularisation properties of ad-MVF in tissue-engineering applications.

Seeding density is a crucial determinant for the in vivo vascularisation capacity of adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments (ad-MVF) represent effective vascularisation units for the seeding of dermal substitutes. However, particularly in case of extensive skin defects, the required amounts of donor fat tissue for the harvesting of ad-MVF may not always be available. Therefore, we herein determined the lowest ad-MVF density needed to induce a sufficient vascularisation and incorporation of seeded implants. Collagen-glycosaminoglycan matrices (Integra®; diameter: 4 mm) were seeded with 15,000 (HD), 10,000 (MD) and 5,000 (LD) ad-MVF and implanted into full-thickness skin defects within mouse dorsal skinfold chambers, to analyse their in vivo vascularisation and incorporation. Intravital fluorescence microscopy showed a comparable vascularisation of HD and MD ad-MVF-seeded Integra®, which was significantly higher when compared to LD ad-MVF-seeded Integra®. As assessed by photoacoustic imaging, this was associated with an increased oxygenation of the implants. Additional histological and immunohistochemical analyses revealed an enhanced cellular infiltration, collagen content, microvessel density and epithelialisation of HD and MD ad-MVF-seeded Integra®, indicating a better incorporation compared to LD ad-MVF-seeded implants. These findings demonstrate that 80,000 ad-MVF/cm² is the least required density to guarantee an effective vascularisation of the dermal substitute.

Potentials and limitations of Integra® flowable wound matrix seeded with adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularisation units for bioengineered Integra® matrix wound dressing (MWD). However, due to the sheet-like structure with small pore sizes, the seeding of this matrix with ad-MVF is mainly limited to its surface. Integra® flowable wound matrix (FWM) may be suitable to achieve a more homogeneous distribution and, thus, improved vascularisation, because this gel-like matrix allows for the direct admixture of ad-MVF during sample preparation. To test this hypothesis, we seeded MWD and FWM with an identical number of ad-MVF and assessed their distribution and inter-fragment distance within both matrices. Moreover, ad-MVF-seeded MWD and FWM were implanted into full-thickness skin defects within mouse dorsal skinfold chambers to analyse their vascularisation, epithelialisation and tissue incorporation using intravital fluorescence microscopy, histology and immunohistochemistry. Seeded FWM exhibited a more homogeneous ad-MVF distribution, when compared to MWD. This resulted in a significantly increased inter-fragment distance, preventing the reassembly of ad-MVF into new microvascular networks. Accordingly, the vascularisation of FWM was diminished after implantation, as indicated by a reduced functional microvessel density and blood perfusion. This was associated with a decreased tissue incorporation and epithelialisation of the matrix, when compared to ad-MVF-seeded MWD. Hence, the use of FWM as a carrier system may require a tremendous amount of ad-MVF to shorten their inter-fragment distance and, thus, to maintain their vascularisation capacity for tissue engineering applications.