Engineered composite tissue as a bioartificial limb graft

Abstract

The loss of an extremity is a disastrous injury with tremendous impact on a patient's life. Current mechanical prostheses are technically highly sophisticated, but only partially replace physiologic function and aesthetic appearance. As a biologic alternative, approximately 70 patients have undergone allogeneic hand transplantation to date worldwide. While outcomes are favorable, risks and side effects of transplantation and long-term immunosuppression pose a significant ethical dilemma. An autologous, bio-artificial graft based on native extracellular matrix and patient derived cells could be produced on demand and would not require immunosuppression after transplantation. To create such a graft, we decellularized rat and primate forearms by detergent perfusion and yielded acellular scaffolds with preserved composite architecture. We then repopulated muscle and vasculature with cells of appropriate phenotypes, and matured the composite tissue in a perfusion bioreactor under electrical stimulation in vitro. After confirmation of composite tissue formation, we transplanted the resulting bio-composite grafts to confirm perfusion in vivo.

Keywords: Bioprosthesis; Bone graft; Mechanical properties; Muscle.

Fig. 1. Perfusion decellularization of isolated forearms

(a) Photograph of an isolated forearm, cannulated through the brachial artery for detergent perfusion, yielding acellular scaffolds after 35 hours. (b, c) Pentachrome stained serial cross-sections of native (left) and decellularized (right) whole forearms at different anatomic levels showing skin (s), tendons (t), bone (b) and muscle (m) tissue (b=mid-forearm, c=mid-hand. Insets show representative artery). (d) Masson’s Trichrome stain of native (left) and decellularized (right) muscle tissue showing artery (a), vein (v), nerve (n) and muscle (m) tissue. (e) Masson’s Trichrome stain of native (left) and decellularized bone showing cortical bone (CB) and medullary cavity (MC) (C) (f, g) Biochemical quantification of DNA (f) and glycosaminoglycan (g) content of native and decellularized muscle tissue. (n ≥ 3 forearms, values are mean ± SD, asterisk indicating P < 0.05). (h–j) 3D microtomography reconstruction of soft tissue and bone in cadaveric (h) and decellularized (i) forearms and corresponding axial cross-sections at the mid-forearm level. White arrows indicating neurovascular bundles. (j) 3D reconstruction of CT-angiography of a decellularized forearm showing integrity of vascular conduits (white arrows).

Fig. 2. Protein content and biomechanical properties

(a–d) Immunohistochemical staining of native (top) and decellularized (bottom) tissue for alpha actinin (a), alpha skeletal myosin (b), collagen IV (c) and collagen X (d). Scale bar, 100μm (e) 3D microtomography was used to analyze cortical geometry. (f–i) Peripheral dual-energy x-ray absorptiometry (pDXA) was performed to analyze bone mineral density and three-point bending analyses to determine mechanical properties after decellularization (n = 3). Error bars are SD. There were no significant differences between decellularized and native bone properties. (j) Passive mechanical testing of the musculoskeletal system before and after decellularization. Passive tendon traction was measured to determine mechanical properties of the passive musculoskeletal system after decellularization (n=4, values are mean ± SD, asterisk indicating P < 0.05). (k) Proteomic analysis of decellularized muscle matrix showing composition of preserved collagens and proteoglycans.

Fig. 3. Regeneration of composite tissue

(a) Schema of the functional bioreactor for electrical and mechanical stimulation. (b) Schema for composite tissue engineering. First, vascular endothelial cells are instilled into the vascular system of acellular composite tissue grafts. Second, myoblasts, fibroblasts and endothelial cells are injected into the muscle compartment on day 2 of whole organ culture. Third, full thickness skin grafts are transplanted onto engineered constructs on day 10 of in vitro culture. (c) Photograph of regenerated composite tissue graft and photograph of cross-sectional area of a regenerated forearm seeded with C2C12 mouse myoblasts, mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVEC), and skin transplantation (Right). Red dotted circles highlighting ulna and radius. Black dashed line is highlighting full thickness skin graft. (d) Masson’s Trichrome stain of a regenerated whole flexor muscle after 20 days of culture. (e) Masson’s Trichrome stain of regenerated muscle tissue surrounding nerve. High-power field showing aligned, multinucleated myofibers. (f) Comparison of myofiber diameter in regenerated, neonatal and adult rat muscle tissue (n ≥ 3 muscles, values are mean ± SD, asterisk indicating P < 0.05). (g–h) Immunofluorescence stain for alpha actinin (g), myosin heavy chain (h), and high-power field of multinucleated, striated myofiber (h, right). (i) Immunofluorescence stain for CD31 in re-endothelialized grafts in longitudinal low-power fields for overview and cross-sectional high power field to show perfusable channels (inset). (j) H&E stain of full thickness skin graft (s) with underlying regenerated muscle tissue (m). (k) Immunofluorescence stain for pan-cytokeratin (red) and myosin heavy chain (green). Green signal of stratum corneum is due to autofluorescence (l) Immunofluorescent stain for CD31 (green) and MHC (red) of regenerated, vascularized muscle. Nuclei counterstained with DAPI. Scale bars 100μm (g,h,i,k); 400μm (j); 500μm (e); 1000μm (d); 5000μm (c, right).

Fig. 4. Functional testing of engineered muscle and transplantation

(a) Photograph of engineered muscle after 16 days of organ culture and corresponding longitudinal section in Masson’s Trichrome stain. Scale bar 2000μm. (b) Representative record of the tetanic contractile response of engineered muscle to pulse electrical field stimulation after 16 days of biomimetic organ culture. (pulse = 20V, 50ms). (c) Specific tetanic forces normalized for cross-sectional area for native (N) and regenerated (R) muscle (n = 4 muscles, values are mean ± SD). (e) Anastomosis of engineered composite tissue graft to the blood supply of adult SD rats. (d) Representative recording of intraoperative pressure curves measured in the radial artery of a engineered forearm graft. (f) X-ray image of recipient showing attached composite tissue graft with intramedullary rod (white arrowhead) and intravascular cuff (white arrow). (g) Photograph of explanted tissue showing re-perfused vascular tree (h) Immunohistochemical stain for CD31 on explanted tissue, showing perfusion of re-endothelialized vascular conduits (CD31, brown) with intravascular red blood cells. Scale bar, 100μm

Fig. 5. Perfusion decellularization of primate extremities

(a) Photograph of isolated primate limb. Dotted line represents incision line for volar fasciotomy. Photograph of primate forearm after 24h of decellularization (middle) and completely decellularized limb graft after 148 hours of perfusion decellularization. Perfusion pressure was maintained at 75 mmHg. (b) Representative H&E stain of the humerus. High-power field showing Haversian canal (HC) and acellular osteocyte-lacunae. (c) H&E stain of the cross-section of acellular muscle (m) with attached tendon (t) (separated by dotted line). High-power field showing a magnification of the same area in Masson’s Trichrome stain (top) and acellular muscle in longitudinal section (bottom). (d) Russell Movat’s Pentachrome stain of skin and subcutaneous tissue. (e) Russell Movat’s Pentachrome of neurovascular bundle showing nerve (n), vein (v) and artery (a) with preserved elastic fibers (black) in the arterial wall. High-power fields are H&E stains of the same section and show absence of nuclei and preservation of ultrastructure with nerve fascicle (f) with endoneurium (en) surrounded by perineurium (pn). Arterial wall showing preserved tunica media (tm) and tunica adventitia (ta) Scale bar: 5 cm (a); 1000μm (b–e, low-power fields); 100μm (b–e, high-power fields)