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The vast majority of patients consider the option of prosthesis, butamputees who suffer from large defects such as bilateral aboveelbow amputations adapt poorly and are usually dependent onothers for personal care and hygiene [6]. As a new approach,

allogeneic handtion signicantlyees and eventuallytained with pros-potentially life-

osuppression poselife-saving recon-onor related riskuppression wouldtreatment optionsrm graft from pa-

tient derived cells would therefore be a valid alternative to allo-geneic grafts. Cellular candidates to regenerate the required tissuessuch as muscle progenitor cells, endothelial progenitor cells, andmesenchymal stem cells can be isolated from patients [12e14].However, engineering of a composite tissue graft of the complexityof a hand or a forearm has been impossible to date due to the lack ofappropriate scaffold materials to support the engraftment ofseveral cell phenotypes and the formation of viable and functionaltissue in its physiologic three dimensional context. A recent report

* Corresponding author. Massachusetts General Hospital, 185 Cambridge Street,CPZN 4700, Boston, MA 02114, USA.

Contents lists availab

Biomat

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Biomaterials 61 (2015) 246e256E-mail address: hott@partners.org (H.C. Ott).1. Introduction

In the United States, over 1.5 million people live with limb loss[1]. Amputation is a severe socioeconomic challenge for most pa-tients, causing emotional trauma equivalent to the loss a familymember [2e4]. Therapeutic options after limb loss include recon-structive surgery using autologous tissue, or the use of prostheticdevices ranging from purely aesthetic prostheses to those with afocus on function [5]. Although current prostheses are technicallyhighly sophisticated devices, they only fulll a minimum of phys-iologic function and many offer less than satisfactory aesthetics [5].

worldwide about 70 patients have receivedtransplants since 1998 [7]. Hand transplantaimproved the quality of life of upper limb amputdemonstrated hand function superior to that obthetics [6,8,9]. However, side effects andthreatening complications of long-term immuna signicant ethical dilemma regarding this nonstructive procedure [5,9e11]. A reduction of dfactors, and elimination of long term immunosallow wider application of such reconstructive[6]. Creation of an autologous, bioarticial foreaReceived in revised form22 April 2015Accepted 30 April 2015Available online xxx

Keywords:BioprosthesisMechanical propertiesMuscleBone grafthttp://dx.doi.org/10.1016/j.biomaterials.2015.04.0510142-9612/ 2015 Elsevier Ltd. All rights reserved.The loss of an extremity is a disastrous injury with tremendous impact on a patient's life. Current me-chanical prostheses are technically highly sophisticated, but only partially replace physiologic functionand aesthetic appearance. As a biologic alternative, approximately 70 patients have undergone allogeneichand transplantation to date worldwide. While outcomes are favorable, risks and side effects of trans-plantation and long-term immunosuppression pose a signicant ethical dilemma. An autologous, bio-articial graft based on native extracellular matrix and patient derived cells could be produced on de-mand and would not require immunosuppression after transplantation. To create such a graft, wedecellularized rat and primate forearms by detergent perfusion and yielded acellular scaffolds withpreserved composite architecture. We then repopulated muscle and vasculature with cells of appropriatephenotypes, and matured the composite tissue in a perfusion bioreactor under electrical stimulationin vitro. After conrmation of composite tissue formation, we transplanted the resulting bio-compositegrafts to conrm perfusion in vivo.

2015 Elsevier Ltd. All rights reserved.a r t i c l e i n f oEngineered composite tissue as a bioart

Bernhard J. Jank b, c, Linjie Xiong b, Philipp T. MoseCurtis L. Cetrulo c, d, David A. Leonard c, d, LeopoldoHarald C. Ott a, c, *

a Division of Thoracic Surgery, Department of Surgery, Massachusetts General Hospital,b Center for Regenerative Medicine, Massachusetts General Hospital, USAc Harvard Medical School, Boston, MA, USAd Transplantation Biology Research Center, Department of Surgery, Massachusetts Genee Massachusetts General Hospital, Division of Burn Surgery, Harvard Medical School, US

journal homepage: www.elscial limb graft, c, Jacques P. Guyette b, c, Xi Ren b, c,ernandez b, Shawn P. Fagan e,

ospital, USA

le at ScienceDirect

erials

ier .com/locate/biomateria ls

seeding we mounted the forearm in the biomimetic stimulationbioreactor. We applied no electrical stimulation in the rst 5 days.

a Grass S48 square pulse stimulator (Grass Technologies). Skintransplantationwas performed as follows. Full thickness skin grafts

Baxter). We maintained the matrix in culture for up 21 days.

expanded until passage 6e10.

teriaof successful clinical implantation of acellular biological scaffoldsinto patients suffering from volumetric muscle loss underlines thehuge potential of this principle for reconstructive surgery [15].

Using perfusion decellularization, we have shown that complexcadaveric organs can be rendered acellular, resulting in nativeextracellular matrix (ECM) scaffolds with intact tissue architecturethat can be repopulated with cells to engineer functional tissue[16,17]. To investigate if these methods can be applied to complexcomposite tissues such as limb grafts, we isolated rodent and pri-mate upper limbs, and perfused these with a sequence of detergentand washing solutions via the native vascular system. Perfusiondecellularization led to the removal of cellular material in allrespective tissue compartments, while retaining the mechanicalproperties of the musculoskeletal system. Repopulation of acellularcomposite tissue grafts with muscle progenitor, endothelial andmesenchymal cells resulted in formation of vascularized, muscle-like tissue within its native histological compartment. To enhancethe formation of functional muscle-like tissue, we cultivatedrepopulated limb grafts in a biomimetic bioreactor system,including vascular perfusion and electrical stimulation. Finally, wetested functionality of engineered muscle in terms of isometricforce measurement and patency of the vascular system by ortho-topic limb transplantation.

2. Materials and methods

2.1. Perfusion decellularization

Research animals were cared for in accordance with theguidelines set by the Committee on Laboratory Resources, US Na-tional Institutes of Health, and Subcommittee on Research AnimalCare and Laboratory Animal Resources of Massachusetts GeneralHospital. Male Sprague Dawley rats (Charles River Laboratories)were euthanized with 100 mg/kg ketamine (Phoenix Pharmaceu-tical) and 10 mg/kg xylazine (Phoenix Pharmaceutical) injectedintraperitoneally. After systemic heparinization (American Phar-maceutical Partners) through the IVC, the dissection of the skin ofthe whole upper limb allowed us to identify the brachial artery, thebrachial vein and the nerve plexus. After dissecting the upper limbfrom the shoulder the brachial artery was cannulated with a pre-lled 25G cannula (Luer Stubs, Harvard/Instech) using a surgicalmicroscope. Fasciotomies were performed before ushing theforearm with phosphate buffered saline (PBS). After ushing with5ml PBS the isolated forearmwasmounted into the organ chamberand perfusion was started with 1% SDS (Sigma) for up to 50 h at aconstant ow perfusion of 1 ml/min. This was followed by deion-izedwater for 1 h and 1 h of perfusionwith 1% Triton-X100 (Sigma).To wash out all debris, antibiotic-containing PBS (100 U/mlpenicillin-G; Sigma, 0.25 mg/ml streptomycin; Sigma and ampho-tericin B; Sigma) was used to perfuse the forearm for 124 h.

2.2. Recellularization of decellularized forearms

After washing with PBS for 124 h, decellularized rat forearmswere removed from the decellularization chamber and mounted ina biomimetic stimulation bioreactor system under sterile condi-tions. Prior to cell seeding, we perfused forearm matrixes with37 C oxygenated C2C12 growth medium for at least 1 h at constantow perfusion of 1 ml/min under standard culture conditions(37 C in 5% CO2). The biomimetic simulation bioreactor contains anorgan chamber, which also serves as the main reservoir, in whichthe decellularized forearm is mounted. The bioreactor works as aclosed-circuit system in which medium is perfused into thebrachial artery by a constant ow pump (Ismatec). At day 0 we

B.J. Jank et al. / Biomaseeded the forearmmatrix with 5 106 HUVECs by gravity infusion2.3.2. MEFsMouse embryonic broblasts were purchased from ATCC and

cultured in DMEM, (Gibco) supplementedwith 10% FBS and 1% Pen/Strep, (Sigma) until passage 4e6 on gelatin coated cell-cultureplastic (BD Biosciences).

2.3.3. HUVECsPrimary human umbilical vein endothelial cells (HUVECs) were

purchased from Lonza and expanded in EBM2 endothelial cellmedia (Lonza) supplemented with EGM-2 bulletkit until passage6e10 on gelatin coated cell-culture plastic (BD Biosciences).

2.4. Electrical stimulation bioreactor

We designed and custom built an electrical stimulation biore-actor based on our perfusion bioreactors. Decellularized forearmswere mounted into the bioreactor by clamping the humeral headinto a tissue clamp (Mueller, Germany), stabilizing the whole limb.Carbon rods were submerged into the culturemedium for electricaleld stimulation of the engineered graft. Electrical pulses weregenerated with a square pulse stimulator (Grass S48, Grass2.3. Cell culture

2.3.1. MyoblastsMouse skeletal myoblasts (C2C12) were purchased from ATCC

and expanded in DMEM (Gibco) supplemented with 10% FBS, and1% HyClone Antibiotic Antimycotic solution (Thermo Scientic) ongelatin coated cell-culture plastic (BD Biosciences). Cells werecultured under standard culture conditions (37 C in 5% CO2). Me-dium changed every other day. To avoid myotube formation, cellswere passaged with 0.05% trypsin/EDTA (cellgro-25052CI) at70e80% conuency. For C2C12 differentiation, we supplementedDMEM (Gibco) with 2% horse serum (Gibco) and 1% antibioticantimycotic solution (10,000 units/ml penicillin, 10,000 mg/mlstreptomycin, and 25 mg/ml Amphotericin B; HyClone). C2C12 werewere harvested from the upper extremities of deceased SpragueDawley rats. After removal of access tissue, full thickness skin graftswere meshed to allow for better attachment and perfusion. Afterwashing in PBS, full thickness mesh grafts were transplanted ontothe engineered constructs using sutures and brin glue (Tisseel,We perfused the forearm with C2C12 growth media from day 0 today 3. We changed the medium every 48 h. On day 3, we switchedthe medium to C2C12 differentiation medium supplemented with3% horse serum and EGM2 bulletkit (Lonza). At day 6 we startedelectrical stimulation by applying 6-ms pulses of 20 V and 1 Hzwith(100 cm H2O) into the brachial artery suspended in 75e100 mlEndothelial Cell Growthmedia (EGM-2 Bulletkit; Lonza). After a 60-min static period to allow for cell attachment, we restarted perfu-sion. For regeneration of muscle tissue we injected a cell mixture of10 106 C2C12 cells, 0.5 106 mouse embryonic broblasts andX 106 HUVECs suspended in 1 ml of growth medium troughtwenty injections in the compartments of the decellularized fore-arm with a 27-G needle and a 1-cc tuberculin syringe. After cell

ls 61 (2015) 246e256 247Technologies).

voxel size. The ROI included the entire outer most edge of the

eriacortex. Images were subjected to Gaussian ltration and segmentedusing a xed threshold of 696 mgHA/cm3. The following variableswere computed: total cross-sectional area (bone medullary area)(Tt.Ar, mm2), cortical bone area (Ct.Ar, mm2), medullary area(Ma.Ar, mm2), bone area fraction (Ct.Ar/Tt.Ar, %), cortical tissuemineral density (Ct.TMD, mgHA/cm3), cortical thickness (Ct.Th,mm), as well as maximum, minimum and polar moments of inertia(Imax, Imin, and J, mm4), which describe the shape/distribution ofcortical bone.

2.6. Peripheral dual-energy x-ray absorptiometry (pDXA)

Peripheral dual-energy x-ray absorptiometry (pDXA, PIXImus II,GE Lunar Corp, Madison, WI) was performed on the bones tomeasure bone mineral density (BMD, g/cm3) and bone mineralcontent (BMC, g).

2.7. Mechanical testing of bone

Radii were mechanically tested in three-point bending using anelectrical force materials testing machine (Electroforce 3230, BoseCorporation, Eden Prairie, MN). The bending xture had a spanlength of 14 mm. The test was performed with the load point indisplacement control moving at a rate of 0.03 mm/s. All of thebones were positioned in the same orientation during testing withthe anterior surface resting on the supports and being loaded intension. Bending stiffness (EI, N-mm2), estimated modulus ofelasticity (E, GPa), estimated bending strength (sult, MPa), andfracture energy (mJ) were calculated based on the force anddisplacement data from the tests and the mid-shaft geometrymeasuredwith mCT. Fracture energy is the energy that was requiredto cause the radius to fracture and it was calculated by nding thearea under the forceedisplacement curve using the Riemann Summethod. Bending stiffness was calculated using the linear portion ofthe forceedisplacement curve. The maximum moment of inertia(IMax) was used when calculating the estimated modulus of elas-(Microl, Flow Tech Inc.) In brief, Microl radiopaque silicone wasinjected into the brachial artery of a decellularized forearm sub-merged in PBS. After polymerization overnight, specimens weredehydrated trough a series of increasing ethanol concentration andcleared by submersion in methyl salicylate (Fisher Scientic).

To assess cortical bone parameters, 100 transverse mCT sliceswere obtained at the femoral mid-diaphysis using a 16 mm isotropic2.5. Microtomography

A high-resolution desktop micro-tomographic imaging system(mCT40, Scanco Medical AG, Bruttisellen, Switzerland) was used toassess cortical bone morphology and mineral density at the mid-shaft of the ulna and radius. Scans were acquired using a 16 mm3

isotropic voxel size, 70 kVP, 114 mAs, 300 ms integration time, andwere subjected to Gaussian ltration and segmentation. Imageacquisition and analysis protocols adhered to the JBMR guidelinesfor the use of mCT in rodents.

To enhance the contrast of decellularized and cadaveric muscletissue, forearmswere stainedwith 0.3% Phosphotungstic Acid (PTA;Sigma). For this purpose, isolated forearms were mounted in aperfusion chamber lled with 0.3% PTA dissolved in 70% ethanoland perfused via the brachial artery for 48 h at room temperature.After staining, the specimens were scanned, mounted in 70%ethanol using a SkyScan 1173 high energy spiral scan micro-CT.

To highlight vascular conduits, angiography was performed on adecellularized forearm graft using a radiopaque silicone cast.

B.J. Jank et al. / Biomat248ticity and bending strength.2.8. Passive tension traction testing

Dissected rat limbs were mounted on a platform using tissueclamps (Mueller, Germany) and a tissue retractor, which allowedmovement only in thewrist. Flexor carpi radialis and Extensor carpiradialis tendons were attached to an isometric force transducer(TRI202PAD, Panlab, Spain) using surgical sutures (6-0 silk), whichwas mounted on a Miniature Dovetail Stage (M-MT-AB2, Newport).Knots (4-0 monolament, Ethicon) were placed in the skin toidentify the radiocarpal and metacarpophalangeal joint. Tensionwas gradually increased from 0 g to 25 g using the micrometerscrews of the Miniature Dovetail Stage and the degree of exionrespectively extension in the wrist was recorded using a digitalcamera (Nikon). Recording of the tension datawas performed usingPower Lab (AD Instruments). Analysis of the footage was done us-ing ImageJ (NIH). Degree of exion respectively extension wasmeasured from the actual resting position of the joint presented asmean with STDEV; Statistical comparison between groups wereperformed using a two-tailored student's t-test; n 4.

2.9. LC-MS/MS Proteomic matrix analysis

Excised gel bands of decellularized muscle were digested withtrypsin. Peptide sequence analysis of each digestion mixture wasperformed by microcapillary reversed-phase high-performanceliquid chromatography coupled with nanoelectrospray tandemmass spectrometry (LCMSMS) on an LTQ-Orbitrap Velos massspectrometer (ThermoFisher Scientic, San Jose, CA). The Orbitraprepetitively surveyed an m/z range from 395 to 1600, while data-dependent MS/MS spectra on the twenty most abundant ions ineach survey scan were acquired in the linear ion trap. MS/MSspectra were acquired with relative collision energy of 30%, 2.5 Daisolation width, and recurring ions dynamically excluded for 60 s.Sequencing of peptides was facilitated with the SEQUEST algorithmwith a 30 ppm mass tolerance against a the rattus norwegicussubset of the Uniprot Knowledgebase supplemented with a data-base of common laboratory contaminants, concatenated to areverse decoy database. Using a custom version of ProteomicsBrowser Suite (PBS v.2.7, ThermoFisher Scientic) peptide-spectrum matches (PSMs) were accepted with mass error

teriarunning tap water (solutions all from Fisher). Premade antigenretrieval solution (Antigen Unmasking Solution H-3300, VectorLaboratories) was used to do antigen retrieval. We heated the slidesin antigen retrieval solution until the temperature reached 95 C for20 min. After antigen retrieval, slides were allowed to cool down toroom temperature. Slides were then washed in PBS at room tem-perature for 5e10 min. After washing in PBS, slides were blockedusing dual endogenous enzyme-blocking reagent (Dako) for 5 min.After incubating the slides in PBS for another 5 min, antigenblocking was performed using 1% BSA (Sigma) in 1x PBS for another30 min. After blocking, primary antibodies were added and incu-bated at 4 C overnight. A humidied chamber was used for allincubation steps. After washing in PBS, secondary antibody wasadded and incubated for 40 min at room temperature. Slides werewashed in PBS for 5 min prior to Diaminobezidine (DAB) devel-opment. After DAB development slides were washed in deionizedwater and counterstained with hematoxylin following standardprotocols. After dehydration, drops of Permount Mounting Media(Fisher Scientic) were added on the slide and covered with acoverslips.

For immunouorescence, tissue sections were deparafnized,rehydrated, and rinsed in PBS with 0.1% Triton X-100 for 15 min.Antigen retrieval was performed with 10 mM sodium citrate, pH6.0, at 95 C for 30 min. Sections were then blocked with 1% BSA inPBS for 1 h at room temperature. Primary antibodies were appliedin blocking buffer overnight at 4 C, followed by secondary anti-bodies at 1:400 dilution for 1 h at room temperature. Secondaryantibodies used were Alexa-Fluor anti-mouse 488 and anti-rabbit594 (Invitrogen). Slides were mounted using DAPI-containingmounting media (Vector Labs), and images acquired using aNikon Ti-E inverted uorescent microscope.

2.12. Isometric force measurement

Isometric force measurement was performed on day 14e16 ofin vitro culture and compared to rat neonatal forearm muscles.Individual native or engineeredmuscle bers (n 4) were attachedto a force transducer (Model # 403A, Aurora Scientic) on one endand at the other end to a stainless steel pin between two carbonelectrodes for electrical eld stimulation. The tissue was suspendedin an organ bath lled with Krebs Henseleit solution (SigmaAldrich). The solution was oxygenated with 100% O2 and main-tained at 37 C using a heating bath circulator (Lauda E100, Ger-many). After applying pretension of 150e180 mg, the tissue wasallowed to equilibrate for 10 min. The maximum contractile forceswere measured at 120 Hz, 60 V, 50 ms duration and 1,5 s trainduration. Electrical stimulation was provided with a Grass S48stimulator (Grass Technologies) and recording of the force mea-surement was performed using Power Lab (AD Instruments).

2.13. Morphometric measurement of myobers

High-powered elds (20 ) were selected from neonatal, adultor regenerated muscle of H&E or IF-stained sections (5 mm) (n 3in each group). Myober diameter was measured using ImageJ(NIH) and statistical analysis was performed using Microsoft Excelfor Mac 2011 (mean SEM). All measurements were averaged pergroup to determine mean values SEM.

3. Results

3.1. Perfusion decellularization of whole limb grafts

We harvested limb grafts from adult SD rats and perfused the

B.J. Jank et al. / Biomatissue via the brachial artery with a 1% sodium dodecyl sulfate(SDS) based protocol. We noticed that tissue edema developedwithdecellularization, which led to increased compartment pressuresand compromised perfusion. By performing fasciotomies beforeinitiation of detergent perfusion, we were able to allow for radialtissue expansion without inhibiting perfusate ow. Since rats areloose skinned animals (their skin is loosely attached to underlyingtissue and can slide and retract extensively), we decided tocompletely remove the skin of the forearm in the rat model. Lowow (0.6e0.8 ml/min) arterial perfusion was maintained withtypical decellularization duration in the range of 24e50 h(33 h 14 h). During the decellularization process, perfusionpressure oscillated between 20 and 185 mmHg. Over the course ofseveral hours of detergent perfusion, the tissues remained intact ongross morphologic examination, while the muscle compartmentsbecame nearly translucent (Fig. 1a, Movie S1). This observationcorresponded to removal of cellular tissue components and pres-ervation of composite tissue architecture and extracellular matrixstructure of muscles, tendons, bones, ligaments, nerves, and bloodvessels on histologic examination (Fig. 1bee and Fig. 2aed). Thehoneycomb patterned ECM on histological cross-section of decel-lularized grafts represent preserved endomysial sheets surround-ing each single muscle ber in native muscle (Fig. 2a,b insets). Axialmicrotomographic imaging conrmed complete preservation ofthree-dimensional composite tissue architecture with decreasedvolume and signal intensity in soft tissues (Fig. 1h,i), but main-tained cortical geometry and tissue mineral density of bone tissue(Fig. 2e,g). Additional analysis using peripheral dual-energy x-rayabsorptiometry (pDXA), and three-point bending test were per-formed and revealed no signicant effect of decellularization onmechanical, mineral or geometric bone characteristics (Fig. 2eei).Thus, no weakening of the skeletal system occurs in decellularizedtissue compared to native tissue. Microtomographic angiographyconrmed presence of perfusable vascular channels throughout theentire graft owing from larger to smaller vessels (Fig. 1j). Passivemechanical testing conrmed functional preservation of the entireskeletomuscular apparatus with intact osteotendinous junctionsand maintained stability and full range of motion in joints of wrist,and digits (Fig. 2j). These ndings are in line with our own expe-rience with decellularization of heart muscle [17], and data ondecellularized tendon [18], nerve [19], ligament-bone [20] andbone grafts [21] by other groups. Proteomic analysis conrmed thepreservation of collagens and glycosaminoglycans within themuscle ECM. (Fig. 2k). Histological analysis and immunohisto-chemical staining demonstrated decellularization of all tissuecompartments (Fig. 1bee), leaving no motor proteins detectable(Fig. 2a,b), while preserving collagens (Fig. 1d,e and Fig. 2c,d).Biochemical analysis showed that our decellularization protocolremoved approximately 90% of the DNA content, while retaining40% of the sulfated glycosaminoglycan (GAGs) content of nativemuscle tissue (Fig. 1f,g). Hence, perfusion decellularization isapplicable to composite tissue and produces acellular limb scaffoldswith preserved anatomical structure and mechanical properties ofthe musculoskeletal system.

3.2. Biomimetic culture

To determine if perfusion decellularized composite tissue ma-trixes could be repopulated with myogenic, vascular, and mesen-chymal cells to form viable grafts, we designed a perfusionbioreactor enabling perfused long-term culture under sterile con-ditions (Fig. 3a). Since electrical stimulation has been described toimprove the formation of functional muscle [22], we includedcarbon electrodes in the bioreactor for electrical eld stimulation(Fig. 3a,A). Culture mediumwas perfused into the brachial artery at

ls 61 (2015) 246e256 249constant ow (1e1.5 ml/min) under standard culture conditions. To

eriaB.J. Jank et al. / Biomat250repopulate composite tissue scaffolds, we infused vascular endo-thelial cell into the brachial artery and injected skeletal myoblastsinto the muscle ECM of selected muscles, and cultured the matrixfor up to 21 days(Fig. 3b). In order to perform skin transplantation,we removed the reseeded constructs from the organ chamber un-der sterile conditions on day 10 and mounted it back after thegrafting procedure to continue perfused organ culture (Fig. 3b).

3.3. Recellularization of acellular composite tissue scaffolds

To regenerate viable composite tissue grafts, we repopulatedacellular composite tissue scaffolds with either C2C12 mouse myo-blasts only (n 7), with a combination of C2C12 mouse myoblastsand mouse embryonic broblasts (n 20) for muscle regeneration,or with a combination of C2C12 mousemyoblasts, mouse embryonicbroblasts and human umbilical vein endothelial cells (HUVEC) forregeneration of re-endothelialized composite tissue (n 5). Finally,

Fig. 1. Perfusion decellularization of isolated forearms. (a) Photograph of an isolated forescaffolds after 35 h (b,c) Pentachrome stained serial cross-sections of native (left) and decel(t), bone (b) and muscle (m) tissue (b mid-forearm, c mid-hand. Insets show represemuscle tissue showing artery (a), vein (v), nerve (n) and muscle (m) tissue. (e) Masson's Trmedullary cavity (MC) (f,g) Biochemical quantication of DNA (f) and glycosaminoglycanmean SD, asterisk indicating P < 0.05). (h-j) 3D microtomography reconstruction of soft ticross-sections at the mid-forearm level. White arrows indicating neurovascular bundles. (j)vascular conduits (white arrows).ls 61 (2015) 246e256we performed skin transplantation onto the engineered compositetissue graft (n 5) (Fig. 3b,c). Regeneration of the different com-partments was done stage by stage with specic subsequent cul-ture periods. (Scheme Fig. 3 b) For repopulation of muscle, wetested two different seeding strategies. Seeding trough vascularinfusion of the cell population at constant high ow over the courseof several hours resulted only in marginally distribution of myo-blasts into muscle ECM with the majority of cells retained in thevascular conduit and therefore appeared not suitable for repopu-lation of muscle tissue. Injection of the cells into the muscle ECMresulted in good engraftment and muscle like tissue formation,however, the resulting mechanical trauma also disrupted ECMstructure, therefore disturbingmicrocirculation and cell integrationinto the matrix, which led to areas of cell apoptosis at the injectionsite. Since vascular resistance increases with decellularization anddecreases with re-endothelialization [23], we rst seeded acellularmatrixes with endothelial cells. Instillation of HUVECs trough the

arm, cannulated through the brachial artery for detergent perfusion, yielding acellularlularized (right) whole forearms at different anatomic levels showing skin (s), tendonsntative artery). (d) Masson's Trichrome stain of native (left) and decellularized (right)ichrome stain of native (left) and decellularized bone showing cortical bone (CB) and(g) content of native and decellularized muscle tissue. (n 3 forearms, values aressue and bone in cadaveric (h) and decellularized (i) forearms and corresponding axial3D reconstruction of CT-angiography of a decellularized forearm showing integrity of

teriaB.J. Jank et al. / Biomabrachial artery followed by 60 min of static culture led to homog-enous lining of the vascular system (Fig. 3i) Engraftment efcacy ofa single seeding step was 69% 15% (n 4).

Next, we seeded C2C12 mouse myoblasts on day two of wholeorgan culture in co-culture with mouse embryonic broblasts or intri-culture with broblasts and HUVECs by injection into the acel-lular muscle matrix of selected muscles. The injection of micro-depots using a surgical microscope resulted in good engraftmentof the cells and eventually led to the formation of functional,muscle-like tissue (Fig. 3deh). Since HUVECs deplete quickly inhigh glucose DMEM based media, we tested different media for-mulations for the maintenance of all cell types. We found lowglucose DMEM supplemented with 10% FBS, 2% HEPES, VEGF, hy-drocortisone, rhFGF-B, rhEGF, R3-IGF, ascorbic acid and penicillin(100 IE/mL) suitable for all cell types.

After cell seeding, we mounted the forearm construct back into

Fig. 2. Protein content and biomechanical properties. (a-d) Immunohistochemical staininmyosin (b), collagen IV (c) and collagen X (d). Scale bar, 100 mm (e) 3D microtomography wa(pDXA) was performed to analyze bone mineral density and three-point bending analysesThere were no signicant differences between decellularized and native bone properties. (larization. Passive tendon traction was measured to determine mechanical properties of theasterisk indicating P < 0.05). (k) Proteomic analysis of decellularized muscle matrix showinls 61 (2015) 246e256 251the bioreactor and started perfusion after 60 min of static culture.Switching to a low-serum differentiation medium induced Myotubefusion. Electrical stimulation during growth and differentiationphase greatly enhanced cell alignment along endomysial sheets andeventually led to the formation of functional muscle tissue(Fig. 4aec). Myober diameter was intermediate between neonataland adult rat muscle tissue after 14 days of in vitro culture (Fig. 3f).

Finally, full thickness skin grafts were harvested from the upperextremities of donor rats and transplanted onto the engineeredcomposite tissue grafts on day 10 of whole organ culture. Skingrafts were attached to the engineered tissue using sutures andbrin glue.

To evaluate the distribution and phenotype of cells in the bio-articial forearm grafts, we performed histological analysis. Seededcell types engrafted in their appropriate compartments (Fig. 3dek).Myoblasts seeded into the acellular muscle matrix fused to

g of native (top) and decellularized (bottom) tissue for alpha actinin (a), alpha skeletals used to analyze cortical geometry. (f-i) Peripheral dual-energy x-ray absorptiometryto determine mechanical properties after decellularization (n 3). Error bars are SD.j) Passive mechanical testing of the musculoskeletal system before and after decellu-passive musculoskeletal system after decellularization (n 4, values are mean SD,g composition of preserved collagens and proteoglycans.

B.J. Jank et al. / Biomateria252multinucleated, striated bers (Fig. 3h, right), aligned along thelongitudinal axis and expressed a-actinin and myosin heavy chain(Fig. 3 g,h), as seen in native muscle tissue. HUVECs seeded into thebrachial artery aligned along the vascular bed to form perfusablechannels and expressed CD-31 (Fig. 3i).

Fig. 3. Regenerationof composite tissue. (a) Schemaof the functional bioreactor for electrical sinstilled into the vascular systemof acellular composite tissue grafts. Second,myoblasts, broblaculture.Third, full thickness skingrafts are transplantedontoengineeredconstructsonday10of insectional area of a regenerated forearmseededwith C2C12mousemyoblasts,mouse embryonicRed dotted circles highlighting ulna and radius. Black dashed line is highlighting full thickness skculture. (e)Masson's Trichrome stain of regeneratedmuscle tissue surroundingnerve. High-powregenerated, neonatal and adult rat muscle tissue (n 3 muscles, values are mean SD, asteriskchain (h), and high-power eld of multinucleated, striated myober (h, right). (i) Immunouooverview and cross-sectional high power eld to show perfusable channels (inset). (j) H&EImmunouorescence stain for pan-cytokeratin (red) and myosin heavy chain (green). Green sig(green) and MHC (red) of regenerated, vascularized muscle. Nuclei counterstained with DAPI. Sls 61 (2015) 246e2563.4. Functional testing of engineered muscle

To determine whether engineered composite tissue graftswould contain functional skeletal muscle, we performed isometricforce measurement on isolated muscles through electrical eldstimulation (EFS) on day 14e16 of in vitro culture (Fig. 4aec). Fig. 4

timulation. (b) Schema for composite tissue engineering. First, vascular endothelial cells arests and endothelial cells are injected into themuscle compartment on day 2 ofwhole organvitro culture. (c)Photographof regeneratedcomposite tissuegraftandphotographof cross-broblasts, humanumbilical vein endothelial cells (HUVEC), and skin transplantation (Right).in graft. (d)Masson's Trichrome stain of a regeneratedwhole exormuscle after 20 days ofereld showing aligned,multinucleatedmyobers. (f) Comparison ofmyober diameter inindicating P < 0.05). (geh) Immunouorescence stain for alpha actinin (g), myosin heavyrescence stain for CD31 in re-endothelialized grafts in longitudinal low-power elds forstain of full thickness skin graft (s) with underlying regenerated muscle tissue (m). (k)nal of stratum corneum is due to autouorescence (l) Immunouorescent stain for CD31cale bars 100 mm (g,h,i,k); 400 mm (j); 500 mm (e); 1000 mm (d); 5000 mm (c, right).

cic force of neonatal muscle (Fig. 4c).

treatments for the loss of composite tissue such as hand amputa-tion are merely palliative. Although many challenges remain, we

of enconfor cf adacheee (hle b

teria3.5. Transplantation of engineered composite tissue grafts

To show that engineered composite tissue grafts can be used forsurgical reconstruction after limb loss, we performed orthotopiclimb transplantations. The native limbwas exposed and amputatedb and c show a representative record of tetanic forces generated byengineered muscle tissue under EFS. The average peak isometricforce was 18,7 mN 1,6 (n 4). The average specic force was105 N/m2 for engineered myober and 135 N/m2 for rat neonatalmuscle and therefore represented approximately 80% of the spe-

Fig. 4. Functional testing of engineered muscle and transplantation. (a) Photographin Masson's Trichrome stain. Scale bar 2000 mm. (b) Representative record of the tetanicof biomimetic organ culture. (pulse 20 V, 50 ms). (c) Specic tetanic forces normalizedmean SD). (e) Anastomosis of engineered composite tissue graft to the blood supply othe radial artery of a engineered forearm graft. (f) X-ray image of recipient showing attcuff (white arrow). (g) Photograph of explanted tissue showing re-perfused vascular trendothelialized vascular conduits (CD31, brown) with intravascular red blood cells. Sca

B.J. Jank et al. / Biomaat the level of the mid-humerus while preserving the recipientsupper arm muscles. Limbs were transplanted onto isogenic SD ratsusing a modied cuff technique for anastomosis of the vasculature.Graft brachial vessels were anastomosed to recipient axillary ves-sels, the brachial plexus approximated end-to-end and osteosyn-thesis was performed using an intramedullary rod (Fig. 4e,f). Bicepsand triceps tendons were re-approximated to the recipient's biceps,respectively triceps. Upon unclamping of the artery, the vascularbed lled with blood (Fig. 4g,h). We inserted a pressure catheter inthe radial artery to conrm pulsatility of blood ow (Fig. 4d). Inaddition to our passive tendon traction testing in vitro, we attemptto transplant a bioarticial hand graft at the level of the distalforearm to conrm preservation of mobility and the functionalpotential of the bioarticial graft in-vivo. After reconnection of thebone, we approximated exor and extensor tendons to the re-cipients forearm muscles. Upon electrical stimulation of the re-cipients forearm muscles, the bioarticial graft performed a exionmovement in thewrist andmetacarpophalangeal joints (Movie S2).

To show that composite tissue perfusion decellularization canbe scaled to clinically relevant size, we decellularized whole pri-mate forearms (Fig. 5) at a constant perfusion pressure of 70mmHgthrough the brachial artery. Similar to our rat experiments, weperformed fasciotomies (Fig. 5a, dotted line) to allow for radialtissue expansion during the decellularization process but we didnot dissect the skin. Histological examination showed the removalof cellular material in bone tissue compartments (Fig. 5b), muscleand tendons (Fig. 5c), the skin and subcutaneous tissue (Fig. 5d) andpresent a rst step towards regeneration of a bioarticial compositetissue graft, by using the forearm as a proof of principle. Prior workneurovascular bundles (Fig. 5e) with preservation of ECM ultra-structure after 7 days of detergent perfusion (Fig. 5a).

4. Discussion

Bioarticial composite tissue grafts, engineered using patientderived cells on demand, could become a tailored treatment op-tion for patients suffering from volumetric tissue loss. Current

gineered muscle after 16 days of organ culture and corresponding longitudinal sectiontractile response of engineered muscle to pulse electrical eld stimulation after 16 daysross-sectional area for native (N) and regenerated (R) muscle (n 4 muscles, values areult SD rats. (d) Representative recording of intraoperative pressure curves measured ind composite tissue graft with intramedullary rod (white arrowhead) and intravascular) Immunohistochemical stain for CD31 on explanted tissue, showing perfusion of re-ar, 100 mm.

ls 61 (2015) 246e256 253in decellularization of isolated tissues such as tendon [24], bone[25], nerve [26], and muscle [27] inspired us to attempt to generateacellular scaffolds containing all of these tissues in their physiologiccontext, while keeping the composite architecture and biome-chanics of a graft as complex as a forearm intact. Moreover, thefeasibility of using decellularized tissue for the treatment of soft-tissue loss was highlighted by a recent report of successful treat-ment of volumetric muscle loss in 5 patients using an acellularxenogeneic ECM scaffold [15]. To accomplish this, we adapted apreviously reported technique developed for heart [17], lung[16,28], liver [29] and kidney [23] taking advantage of the nativevasculature to deliver decellularization agents, hence allowing forintense penetration of the agents into the tissue compartments.Consistent with the collective experience with solid organs, wefound that detergent decellularization of whole rat and baboonextremities removes cellular components throughout the entiregraft and its diverse tissue compartments. Although it is hardlypossible to remove all cellular components and nuclear fragmentscompletely from a tissue [30], removal of DNA to an adequately lowlevel is vital for avoiding adverse immune reaction. We conrmeddecellularization of composite tissue grafts by biochemical andhistological analysis. In our experiments, DNA content decreased byapproximately 90 % to 350 ng/mg tissue, which is comparable toresidual DNA content of decellularized kidney [31] and esophagus[32] in other studies.

Notably, a specic threshold amount of residual DNA that wouldfacilitate a proinammatory response against the scaffold is still

B.J. Jank et al. / Biomaterials 61 (2015) 246e256254unknown.[30] Moreover, other studies have shown that decellu-larized tissues with comparable residual DNA content such asesophagus [32] or liver tissue, decellularizedwith a similar protocol[33], are well tolerated in in-vivo implantation experiments and noadverse immune reactions are induced. Decellularization not onlyremoves cellular components, but also causes a collateral loss ofECM components. Loss of ECM components increases withextended duration of decellularization [34], which subsequentlyalters the biomechanical properties and biochemical compositionof engineered grafts. We therefore aimed to keep the decellulari-zation process as short as possible, by allowing for a higherperfusion pressure; therefore, we were able to retain 40% ofsulfated glycosaminoglycans in our experiments, which is in linewith results of other studies [31].

Retaining the biomechanical properties of the composite tissuescaffold as well as the range of motion of joints during the decel-lularization process is a requirement for its function upon regen-eration and dependent on the preservation of ECM componentsduring decellularization. We performed Micro-computed tomog-raphy, peripheral dual-energy x-ray absorptiometry, and three-

Fig. 5. Perfusion decellularization of primate extremities. (a) Photograph of isolated pprimate forearm after 24 h of decellularization (middle) and completely decellularized limb75 mmHg. (b) Representative H&E stain of the humerus. High-power eld showing Haveracellular muscle (m) with attached tendon (t) (separated by dotted line). High-power eld shmuscle in longitudinal section (bottom). (d) Russell Movat's Pentachrome stain of skin and snerve (n), vein (v) and artery (a) with preserved elastic bers (black) in the arterial wall. Hpreservation of ultrastructure with nerve fascicle (f) with endoneurium (en) surroundedadventitia (ta) Scale bar: 5 cm (a); 1000 mm (b-e, low-power elds); 100 mm (b-e, high-popoint bending analysis to evaluate the biomechanical propertiesof decellularized bone within the composite tissue graft. We foundthat perfusion-decellularization of the graft did not signicantlyaffect the mechanical, mineral, or geometric characteristics of bonetissue. In order to perform movement, joints of the engineeredcomposite tissue graft must stay exible with a similar ease andrange of motion compared to native joints. We performed a passivetension traction testing and found similar exibility compared tothe native tissue, suggesting that the decellularization had nonegative inuence on joint mobility.

In this initial work, we show that composite tissue scaffold canbe recellularized with a mixture of myogenic, vascular, andmesenchymal cells by seeding each compartment separately. Whilecell seeding of perfusable compartments like the vascular systemcan be accomplished by surface attachment of cells through infu-sion, resulting in a homogeneous distribution, seeding of densetissues without physiological access poses numeral challenges. In-jection of cells using small diameter cannulas under a surgicalmicroscope allowed for targeted cell delivery to distinct musclesheaths and enabled formation of contractile muscle with a similar

rimate limb. Dotted line represents incision line for volar fasciotomy. Photograph ofgraft after 148 h of perfusion decellularization. Perfusion pressure was maintained atsian canal (HC) and acellular osteocyte-lacunae. (c) H&E stain of the cross-section ofowing a magnication of the same area in Masson's Trichrome stain (top) and acellularubcutaneous tissue. (e) Russell Movat's Pentachrome of neurovascular bundle showingigh-power elds are H&E stains of the same section and show absence of nuclei andby perineurium (pn). Arterial wall showing preserved tunica media (tm) and tunicawer elds).

[30] S.F. Badylak, Decellularized allogeneic and xenogeneic tissue as a bioscaffoldfor regenerative medicine: factors that inuence the host response, Ann.

teriamorphology compared to native myober. In order to maintainviability of an engineered composite tissue graft, immediatereperfusion upon transplantation is necessary. We performed CT-angiography to highlight preserved vascular conduits after decel-lularization, which serve as a prerequisite for re-endothelializationand reperfusion in vitro. After seeding of endothelial cells andmaturation in vitro, we observed the formation of perfusablevascular channels, which could withstand physiologic perfusionpressures upon transplantation.

In this work, we met numerous milestones towards the gener-ation of a biomechanically intact composite tissue graft; however,there are several challenges that must be addressed from a trans-lational point of view.

First and foremost, neuronal ingrowth and integration into therecipient's nervous system is a prerequisite to gain ultimate func-tionality of an engineered limb graft.

Equal to allogeneic hand transplantation, axons of the re-cipient's nerve stumps have to regrow into the graft's nerve sheetsin order to reinnervate sensory organs and muscles [35,36].Therefore, peripheral nerve regeneration can only be achievedupon in-vivo implantation.

Restoration of intrinsic handmuscle function in hand transplantrecipients shows that regeneration of neuromuscular junctions in awhole limb is possible [7]. Moreover, regeneration of sensorynerves, resulting in restoration of discriminative sensation, can beobserved in the same patients [7]. Notably, decellularized nervegrafts are already in clinical use to bridge nerve gaps in sensory,motor and mixed nerves [26]. However, such grafts are limited tobridging only short gaps in peripheral nerves. From our point ofview, this might be due to their avascularity and the lack of supportcells and growth factors. In support of this notion, recent studieshave shown that vascularized nerve grafts are superior in restoringfunction compared to free nerve grafts [37,38]. A bioarticialcomposite tissue graft offers preserved neurovascular bundleanatomy, which might allow for neuronal ingrowth similar toallogeneic transplants. Long-term survival experiments will berequired to enable peripheral nerve regeneration, and to ultimatelyproof whether regenerated composite tissue grafts can becomefully functional.

Second, injection of myoblasts in-vitro generates a considerableamount of matrix disruption, therefore disturbing microcirculationand cell integration into the matrix, which leads to areas of cellapoptosis at the injection site. The use of primary or iPS-derivedmesangioblasts, which could be delivered systemically into an ar-tery to regenerate muscle tissue [39], might be a promising cellcandidate for in-vitro muscle regeneration prior to transplantation.Third, we transplanted engineered grafts only in short-term, non-survival procedures. We found that survival procedures at this earlystage of limb regeneration would require extensive postoperativecare, which is hardly possible in a rodent and would furthermoreraise ethical concerns regarding animal welfare.

5. Conclusion

Our results demonstrate the feasibility of producing a complexwhole limb scaffold containing preserved passive musculoskeletalapparatus, vasculature, and nerve sheets, which can be repopulatedwith cells of appropriate phenotype and orthotopically trans-planted into a recipient. In contrast to solid organ transplants,allogeneic composite tissue grafts such as hand transplants are notfully functional at the time of transplantation. Recipient nerveshave to regrow into the donor nerve sheaths, which serve as merescaffold for the recipient's axons, to ultimately reinnervate themuscles and sensory organs within the skin [36]. Bioarticial

B.J. Jank et al. / Biomacomposite tissue grafts may be transplanted at an early stage ofBiomed. Eng. 42 (Jul, 2014) 1517.[31] J.P. Guyette, et al., Perfusion decellularization of whole organs, Nat. Protoc. 9

(2014) 1451.regeneration, and similar to allografts benet from in vivo regen-eration to enable further functional maturation.

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B.J. Jank et al. / Biomaterials 61 (2015) 246e256256

Engineered composite tissue as a bioartificial limb graft1. Introduction2. Materials and methods2.1. Perfusion decellularization2.2. Recellularization of decellularized forearms2.3. Cell culture2.3.1. Myoblasts2.3.2. MEFs2.3.3. HUVECs

2.4. Electrical stimulation bioreactor2.5. Microtomography2.6. Peripheral dual-energy x-ray absorptiometry (pDXA)2.7. Mechanical testing of bone2.8. Passive tension traction testing2.9. LC-MS/MS Proteomic matrix analysis2.10. Histology2.11. Immunohistochemistry2.12. Isometric force measurement2.13. Morphometric measurement of myofibers

3. Results3.1. Perfusion decellularization of whole limb grafts3.2. Biomimetic culture3.3. Recellularization of acellular composite tissue scaffolds3.4. Functional testing of engineered muscle3.5. Transplantation of engineered composite tissue grafts

4. Discussion5. ConclusionReferences