Material-Induced Venosome-Supported Bone Tubes

Abstract

The development of alternatives to vascular bone grafts, the current clinical standard for the surgical repair of large segmental bone defects still today represents an unmet medical need. The subcutaneous formation of transplantable bone has been successfully achieved in scaffolds axially perfused by an arteriovenous loop (AVL) and seeded with bone marrow stromal cells or loaded with inductive proteins. Although demonstrating clinical potential, AVL-based approaches involve complex microsurgical techniques and thus are not in widespread use. In this study, 3D-printed microporous bioceramics, loaded with autologous total bone marrow obtained by needle aspiration, are placed around and next to an unoperated femoral vein for 8 weeks to assess the effect of a central flow-through vein on bone formation from marrow in a subcutaneous site. A greater volume of new bone tissue is observed in scaffolds perfused by a central vein compared with the nonperfused negative control. These analyses are confirmed and supplemented by calcified and decalcified histology. This is highly significant as it indicates that transplantable vascularized bone can be grown using dispensable vein and marrow tissue only. This is the first report illustrating the capacity of an intrinsic vascularization by a single vein to support ectopic bone formation from untreated marrow.

Keywords: angiogenesis; axial vascularization; bioceramic; bioinorganic; material–host interactions; osteogenesis.

Figure 1

A) CAD design of cylindrical implant, B) schematic of the control placement next to femoral arteriovenous bundle and the experimental group with a single femoral vein placed axially within implant. C) Micro‐CT cross‐sections of decalcified implants perfused with MicroFil showing the blood vessels inside and outside the implant (green arrow: fibrous capsule; blue arrow: feeding vein) and quantification of the relative volume of blood vessels inside the implant.

Figure 2

A) Marrow retaining scaffold design and dimensions and positioning of vessels with perforated plastic retainer. B) Marrow aspiration procedure and C–E) scaffold assembly in plastic retainer.

Figure 3

A) Backscattered SEM axial sections from the top, middle, and bottom of scaffolds with high magnification regions. Scale bars: 1 mm and 100 µm, for the low and high magnification, respectively. Significant (P < 0.0001) differences between bone volume and residual cement areas beween groups were observed. B) Microstructure of construct (H&E stained axial cross‐section), left, without intrinsic venous perfusion, new bone (pink) on surface of cement (dark brown and white) with interstitial fibrous tissue; right with induced venosome, significant and contiguous bone entombing residual cement. Bone was visible inside the implant of the vein group (e.g., H&E staining). TRAP positive staining detected only in sample with induced venosome (white arrows).

Figure 4

Representative mineralized axial sections from middle region of the control (left) and the experimental (right) scaffolds at low and high magnification (basic fuchsin and methylene blue). Zones illustrating the ceramic biodegradation are highlighted by the black arrows. Lamellar bone could be observed in the zones delineated by dotted lines, representing high magnification images shown in insets. The 4 and 5 branch stars indicate biocement and bone, respectively. Scale bars on low and high magnification represent 1 mm and 100 µm, respectively. Sections of replicate sample are shown in Figure S2 in the Supporting Information.

Figure 5

Decalcified histology and immunohistochemistry of the control marrow construct, showing top to bottom: H&E at low and high magnification showing the distribution and organization of the extracellular matrix outside and within the scaffold (star = polymer clip, blue arrow = thick vascular capsule surrounding the clip, and yellow arrow = thin vascular capsule surrounding the implant); the expression of α‐smooth muscle actin (brown); type‐IV collagen distribution (in brown). Scale bars on low and high magnification represent 1 mm and 100 µm, respectively.

Figure 6

Decalcified histology and immunohistochemistry of the experimental group (marrow + vein perfusion), showing top to bottom: H&E at low and high magnification showing the distribution and organization of the extracellular matrix outside and within the scaffold (star = polymer clip and blue arrow = thick vascular capsule surrounding the clip); the expression of α‐smooth muscle actin (displayed in brown); type‐IV collagen distribution (in brown). Scale bars on low and high magnification represent 1 mm and 100 µm, respectively. Contrast absence of collagen IV staining inside the scaffold with the nonperfused sample in Figure 5.