Engineering axially vascularized bone in the sheep arteriovenous-loop model

Abstract

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)-loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein-2 (rhBMP-2), were harvested and directly autotransplanted in combination with β-tricalcium phosphate-hydroxyapatite (β-TCP-HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP-2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the β-TCP-HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro-computed tomography (μCT) imaging confirmed the dense vascularization arising from the AV-loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP-2 stimulation in about 12 weeks in the sheep AV-loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies.

Autologous serum improves bone formation in a primary stable silica-embedded nanohydroxyapatite bone substitute in combination with mesenchymal stem cells and rhBMP-2 in the sheep model

“…In contrast, in well vascularized areas, high amounts of carbon and low levels of silicon could be demonstrated. As described in previous studies, 13,15,23,31 a matrix change could be detected particularly near the surface of the bone substitute and only in well vascularized areas. 25 In our constructs, a matrix change was visible in all experimental groups and therefore paves the way for further degradation of the nano-HA bone substitute by osteoclasts and new bone formation.…”

“…The animals were housed in the veterinary care facility under standardized conditions of 55% air humidity and 18°C room temperature with a 12-hour light/dark rhythm as described previously. 23 The sheep were fed once a day with a standard sheep diet (ssniff-Spezialdiäten GmbH, Soest, Germany) and hay and water ad libitum. The animals were deprived of food for 24 hours prior to surgery to limit regurgitation.…”

“…The bone marrow was diluted with phosphate-buffered saline 1:4 (Dulbecco 1×; Biochrom AG, Berlin, Germany) and bone marrow stem cells were harvested by Ficoll gradient centrifugation as described previously. 23 MSC were immediately retransplanted without any interim expansion or storage procedure, or expanded in Dulbecco's Modified Eagle's Medium with 1% penicillinstreptomycin and 10% fetal calf serum (all from Biochrom AG) to a final cell number of 6 million per implant. Before implantation, the cells were labeled using dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate; CellTracker™ CM-DiI, (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.…”

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

“…OCT is an interferometry technique using broadband near-infrared (NIR) light beyond 800 nm. It enables non-invasive and label-free real-time three-dimensional imaging [22,53,54] and relatively deep penetration into tissues (up to 2000 mm) compared with other visible or NIR light techniques ( [39]; table 1). In relation to MRI and mCT, penetration is low; however, what makes OCT strong is that it resolves structures almost at the cellular range.…”

The Arteriovenous Loop: Engineering of Axially Vascularized Tissue

“…A lot of different bone substitutes were implanted, including stable [31] or non-stable scaffolds [30,[32][33][34] composed of 60% hydroxyapatite (HA) and 40% β-tricalcium phosphate (β-TCP) [30][31][32][33][34] or 100% β-TCP [35], natural coral scaffolds [36], processed bovine cancellous bone [37], or nanocrystalline HA in a silica gel matrix [29,38]. To functionalize the scaffolds and to support bone formation, cells can be combined with the replacement material, e.g., non-differentiated bone-marrow derived mesenchymal stem cells (MSCs) [30,32,34,39] or fully differentiated osteogenic cells such as osteoblasts [40]. MSCs are often used in tissue engineering studies due to their multipotent differentiation capacity, their self-renewal and secretion of various growth factors that stimulate angiogenesis, recruitment of other cells, and further regulate the immune response.…”