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Tissue-engineered autologous grafts for facialbone reconstructionSarindr Bhumiratana,1 Jonathan C. Bernhard,1 David M. Alfi,2 Keith Yeager,1 Ryan E. Eton,1

Jonathan Bova,3 Forum Shah,4 Jeffrey M. Gimble,4,5 Mandi J. Lopez,3

Sidney B. Eisig,2 Gordana Vunjak-Novakovic1*

Facial deformities require precise reconstruction of the appearance and function of the original tissue. Thecurrent standard of care—the use of bone harvested from another region in the body—has major limitations,including pain and comorbidities associated with surgery. We have engineered one of the most geometricallycomplex facial bones by using autologous stromal/stem cells, native bovine bone matrix, and a perfusion bio-reactor for the growth and transport of living grafts, without bone morphogenetic proteins. The ramus-condyleunit, the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalizedapproach in skeletally mature Yucatán minipigs (human-scale preclinical model). We used clinically approveddecellularized bovine trabecular bone as a scaffolding material and crafted it into an anatomically correctshape using image-guided micromilling to fit the defect. Autologous adipose-derived stromal/stem cells wereseeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immaturebone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure,integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration thaneither nonseeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility offacial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents aplatform for personalized bone tissue engineering.

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INTRODUCTION

Bone defects in the head and face due to congenital abnormalities,trauma, or cancer surgery are associated with major functional, soci-ological, and psychological problems. Defects in the complex struc-tures of the head and face are difficult to restore with aesthetic andfunctional accuracy. Bone autografts are the gold standard for crani-ofacial reconstruction, owing to their osteoinductive, osteoconductive,and immunocompatible properties (1, 2). However, autografts arelimited in volume, associated with donor site morbidity, and are dif-ficult to carve into precise anatomical shapes. Ideally, the craniofacialbones should be reconstructed to the original anatomy and using thepatient’s own cells.

Stem cell and tissue engineering approaches have made major stridestoward growing biological substitutes that mimic the properties of na-tive tissues. Clinically available bone grafts, either biological or synthet-ic, fall short of providing the fidelity of bone autografts, necessitatingthe development of new treatment modalities. Bone tissues grownfrom culture-expanded stem/progenitor cells isolated from bone mar-row or adipose tissue aspirates are now at the forefront of regenerativemedicine (3–5). Enhanced bone regeneration by transplantation ofvarious types of stem cells has been demonstrated in small-animalmodels (6, 7). Other meritorious studies developed clinically sized an-atomical scaffolds for bone reconstruction by growth factor delivery(8). Early on, imaging data were used in a single patient to create a

1Department of Biomedical Engineering, Columbia University, 500 West 120th Street,New York, NY 10027, USA. 2Division of Oral and Maxillofacial Surgery, Columbia Uni-versity College of Dental Medicine, 630 West 168th Street, New York, NY 10032, USA.3School of Veterinary Medicine, Louisiana State University, Skip Bertman Drive, BatonRouge, LA 70803, USA. 4LaCell LLC, 1441 Canal Street, New Orleans, LA 70112, USA.5Center for Stem Cell Research and Regenerative Medicine, Tulane University Schoolof Medicine, 1324 Tulane Avenue, SL-99, New Orleans, LA 70112, USA.*Corresponding author. Email: gv2131@columbia.edu

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titanium mesh that was filled with bone mineral, bone morphogeneticprotein (BMP), and the patient’s bone marrow; left for 7 months un-der a dorsal muscle to vascularize; and used to fit a mandible defect(9). Scaffolds with stem cells and growth factors were studied in large-animal models, with growth factors alone or in combination with cellsgenerally giving better outcomes (10–13).

However, translational studies in large animals of autologous boneengineered in vitro to precisely match the desired anatomy have notbeen conducted. The complexities associated with growing clinicallysized, living bone grafts have been recognized (14, 15). We previouslydeveloped an anatomically correct scaffold-bioreactor system for bonetissue engineering using osteogenically differentiated mesenchymalstem cells (MSCs) (16). Additive manufacturing of tissue constructshas also been implemented in the repair of long bones (17). Ourmethod is distinctly different as it engineers in vitro geometricallycomplex bones using autologous stem cells, anatomically shaped bonescaffolds, and a geometrically matched bioreactor chamber.

Here, we move engineered bone tissues closer to humans by creat-ing personalized scaffolds to heal critical-sized, load-bearing maxillo-facial bone defects in skeletally mature large animals, the Yucatánminipigs. The investigational approach was designed to provide clin-ical adequacy for repairing the ramus-condyle unit (RCU). We useddecellularized bovine trabecular bone as a scaffolding material, becauseit is clinically approved for bone grafting (Cancello-Pure and CopiOsbone wedges), and crafted it into an anatomically correct shape to per-fectly fit the defect by using image-guided micromilling. Autologousadipose-derived stromal/stem cells (ASCs) were the cells of choice dueto their high availability and easy harvest when compared to bonemarrow–derived cells (MSCs), along with osteogenic capacity (5, 18).The term “autologous” in this study refers to the use of cellular mate-rial prepared from a fat aspirate of the recipient animal and seededinto a cell-free bone matrix.

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In addition to demonstrating feasibility and efficacy of bone repair,we recapitulated the logistics of the envisioned clinical application,where the patient and the manufacturing site are at remote locations,requiring shipping of both adipose tissues for cell isolation and bonegrafts for implantation. The perfusion bioreactor was designed to al-low the growth and transport of bone grafts in a single step whilemaintaining sterility and viability. Thus, from start to finish, the studydemonstrated utility and translation of personalized tissue-engineeredbone grafts for facial reconstruction.

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RESULTS

Bioreactor design for engineering anatomically shapedliving bone implantsOur overarching objective was to grow bone grafts with the preciseanatomical structure to repair a large defect in the jaw, without using

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BMPs or other growth factors; instead, we used only native bonematrix to induce osteogenic differentiation of ASCs through itscomposition, architecture, and mechanical properties (7, 19, 20). Wereconstructed the most eminent weight-bearing facial bone, the RCU,in Yucatán minipigs because of similarities in the jaw anatomy, me-chanics, and bone remodeling to those in humans.

We obtained the bone matrix by removing all cellular materialfrom the native trabecular bone of bovine distal femur, and then fab-ricated the bone matrix into an exact anatomic replica of the nativetissue structure (Fig. 1A). To this end, we used computer-aided micro-milling fabrication methods that were guided by three-dimensional(3D) reconstructions of computed tomography (CT) images of eachpig’s jaw. Autologous porcine ASCs (pASCs) were then cultured for3 weeks in anatomically shaped scaffolds customized to each animal inperfusion bioreactors (movie S1).

Tomaintain cell viability within these complex anatomical scaffolds,we devised an advanced bioreactor system that provided environmental

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Fig. 1. Fabrication of engineered bone grafts. (A) Personalized bonetissue engineering process. Autologous MSCs (from fat aspirates) and CT

casing. The PDMS block was designed as an impression of the anatom-ical RCU structure and contained flow channels at both sides for the

images were obtained for each animal subject. The anatomical scaffoldwas fabricated from the bovine stifle bone that had been processed toremove any cellular material while preserving the tissue matrix. The cellswere seeded into the scaffold and cultured in the specially designedperfusion bioreactor. After 3 weeks, the engineered RCU was implantedback into the pig for 6 months. (B) The bioreactor culture chamber con-sisted of five components designed to provide tightly controlled perfu-sion through the anatomical scaffold: anatomical inner chamber withbone scaffold, two halves of the polydimethylsiloxane (PDMS) (elasto-mer) block with incorporated channels, two manifolds, and an outer

flow of culture medium in and out of the scaffold. The flow rate neces-sary for providing nutrient supply and hydrodynamic shear was definedby the design of parallel channels in the elastomer block (with thechannel diameters and distribution dictated by the geometry of thegraft) and the fluid-routing manifold. The channel diameters andspacing were specifically designed by flow simulation software for eachpig to provide a desired interstitial flow velocity for a given shape andsize of the anatomical RCU. (C) Flow simulation of the medium flowthrough the anatomically shaped scaffold reveals uniform flow velocitythroughout the volume of the graft.

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control and the exchange of nutrients,oxygen, and metabolites. The anatomicalscaffold was placed into the matchingvoid between two elastomer blocks con-taining arrays of channels designed onthe basis of the transport analysis. Theelastomer blocks and the fluid-routingmanifolds were aligned and tightenedby the polycarbonate case to create awatertight seal (Fig. 1B). The perfusion

system distributed culture medium throughout the ~12-ml volumeconstruct by regulating the fluid flow path resistance. Fluid distributioninto the channels was directed by a fluid-routingmanifold via a peristal-tic pump, with one inlet and one outlet per bioreactor. Gas exchangeoccurred within the medium reservoir to maintain oxygen and pH.The medium flow velocity at any specific location within the culturedgraft was controlled by the size of the channels that directed the fluidinto and out of the anatomical chamber (Fig. 1C). For each animal, thechannel sizes (0.82 to 2.41mm) and channel locations were determinedusing computational flow simulation. The interstitial flow velocity ofculture medium was maintained within the cultured grafts between

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400 and 1000 mm/s (Fig. 1C) on the basis of a previously determinedoptimum flow rate that did not limit oxygen transport to the cells (21).

Osteogenic differentiation of adipose stem cells anddeposition of bone matrixAfter 3 weeks of osteogenic differentiation in 2D culture, ASCs fromsubcutaneous porcine adipose tissue exhibited bone cell properties, in-cluding the deposition of alkaline phosphatase (ALP) and mineralizedmatrix (Fig. 2, A and B). After 3 weeks of osteogenic culture in the 3Dscaffolds, the cellularity of the grafts increased 7.5-fold relative to day 3(Fig. 2C). Differentiated ASCs in the engineered bone grafts at week 3

Fig. 2. Properties of engineered bonegrafts. (A) DNA, ALP, and calcium (Ca) con-

tent over the course of 3weeks of osteogen-ic induction. Significant increases in ALP andCa deposition were observed. Data werequantified for each animal as means ± SD(n = 7 animals) using one-way analysis ofvariance (ANOVA) and Tukey’s comparisonposttest. (B) ALP and von Kossa stains con-firmed deposition of ALP protein andminer-al during osteogenic induction. (C) DNAcontent quantifies the number of cells perscaffold at 3 days and 3 weeks after osteo-genic induction in the scaffold-bioreactorsystem, as well as after transport (one addi-tional day). Data are box plots (n = 7). Pvaluesweredeterminedbyone-wayANOVAwith Tukey’s multiple comparison post hoctest. (D) Gene expression after 3weeks of os-teogenic induction of cells in the scaffold.Data are the fold difference (means ± SD)of gene expression normalized to thehousekeeping gene (Gapdh) and then tothe corresponding day 3 data (n = 7). Pvalues were determined by t test with H0 =1. (E) H&E staining and immunohisto-chemistry (type I collagen and BSP) of engi-neered bone grafts after seeding (3 days)and before implantation (3 weeks). Cellsattached to the scaffold (black arrowheads),proliferated, filled the pore spaces, anddeposited bone proteins (white arrow-heads). The decellularized trabecular scaf-fold (S) was maintained throughout thecultivation period. Scale bars, 250 mm.

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expressed osteoblastic transcription factors, such as core-bindingfactor a1 (Cbfa1, also known as Runx2) and Osterix; osteogenic matrixproteins Alp, type I collagen (Col1a1), bone sialoprotein (Bsp), andosteonectin (ON); and osteogenic-related growth factors such asBmp2 and transforming growth factor–b2 (Tgfb2), normalized to in-ternal Gapdh control and compared with day 3 (Fig. 2D). The ASCsattached to the bone scaffold surfaces, proliferated over 3 weeks ofcultivation, and filled the pore spaces (Fig. 2E). The cells and thenew osteogenic extracellular matrix (ECM) rich in type I collagenand BSP were present throughout the graft interiors (Fig. 2E).

During graft transport to the site of implantation, the perfusionwas discontinued for up to 10 hours. Finite element analysis of O2

transport and consumption within 3D-engineered bone grafts demon-strated that theminimalO2 concentration at 10hourswas≥0.14mol/m3

(movie S2), a value well above the depletion rate for cell survival. Totest for the effects of reduced oxygen supply on cell viability, we con-ducted experiments with native osteochondral explants of a compara-ble thickness and showed that the cell viability did not change over alonger period of time (96 hours). The average percentage of live cells(±SD) determined by a live-dead assay was 85 ± 12 for the freshlysectioned samples and 83 ± 6 after 96 hours of culture without medi-um perfusion. Consistently, the graft cellularity posttransport was notsignificantly different from the pretransportation values (Fig. 2C).

Pig RCU reconstruction model and bone regenerationFourteen skeletally mature Yucatán minipigs (>2 years old) were cho-sen as study subjects. The left RCU was excised (3 cm wide along thedorsal plane by 6 cm long along the transverse plane), with the rightRCU left intact. The defects were fitted with a tissue-engineered bonegraft or with a cell-free bone scaffold, or left untreated (condylectomy,to control for spontaneous regeneration). The animals were not im-mobilized to maintain native loading environment. There were nocomplications throughout the 6-month study. Animals receiving acel-lular scaffolds and tissue-engineered bone were sacrificed at 3 months(n = 2 each) or 6 months (n = 4 each). Condylectomy subjects weresacrificed at 6 months (n = 2). The left temporomandibular joint(TMJ) capsule was excised with the base of the ramus and temporalbone intact. Engineered grafts had the exact anatomical shape of thenative RCU extracted at the time of surgery (fig. S1).

Bone regeneration and condyle height. CT images of the por-cine skulls were acquired immediately after surgery and at 6 weeks,3 months, and 6 months after implantation (Fig. 3A and fig. S2). Thebone volume (Fig. 3B) and the bone volume fraction (Fig. 3C) in thetissue-engineered bone groups were significantly higher than in the acel-lular scaffold and condylectomy groups at all time points. The greateramount of bone formed in the engineered grafts resulted in a morecomplete regeneration of the original joint anatomy, as indicated bythe final heights of the new condyles (Fig. 3D). The extracted RCUwas about 60% of the original height in all groups. After condylectomy,the condyle height reached about 80% of the structure height. Becausethe geometry and anatomical features of bone grafts matched the nativeRCU, the condyle height after implantation was equivalent to thephysiologic height. Although the condyle height in the acellular scaf-fold group decreased over time, the condyle height in the engineeredbone graft was maintained throughout the study (Fig. 3D).

Ramus regeneration. Regeneration of the ramus bone was ini-tiated from the native bone and progressed toward the grafting region(Fig. 4A and fig. S2). Time lapse of CT images of the individual

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subjects revealed infiltration from the native bone at the rostral endand ossification at the caudal end of the ramus (Fig. 4A and fig.S2). Condylectomy resulted in incomplete ramus regeneration, witha large hollow area within the ramus observed by CT at 6 months aftersurgery (fig. S2). Histological analysis of the condylectomy controlgroup revealed that the region lacking mineralized matrix was filledwith soft fibrous tissue (Fig. 4B and fig. S3). The native and new bonecould be distinguished by the presence of osteocytes that could only beobserved in the newly formed bone.

In contrast to large amounts of acellular scaffold resorption as earlyas 3 months after implantation (fig. S4), tissue-engineered bone graftswere gradually and orderly replaced by new bone, resulting in regen-eration of the original tissues (fig. S5). Portions of both the implantedacellular scaffold and engineered bone were still present at 6 monthsafter implantation (Fig. 4B and figs. S3 to S5 and S7 to S9), indicativeof ongoing remodeling, with remarkable differences in the compo-sition, structure, and bone formation between the two groups.

Condyle regeneration. Regeneration of the condyle could be seenin all groups at 6 months after surgery (Fig. 4C and fig. S6). Longitu-dinal CT images of the individual subjects over 6 months revealed thepatterns of condyle formation and ramus regeneration (fig. S6). In thecondylectomy group, ossification occurred in the condyle region andgrew in size over time, forming a condyle structure supported by anarrow ramus at the rostral and caudal ends (Fig. 4C). At 6 months,the condylar cartilage had not yet regenerated, resulting in under-developed condylar surfaces (Fig. 4D and fig. S7).

Acellular scaffold implantation resulted in new tissue at the artic-ular surfaces of the graft material. By 3 months, the new bone formedat the articular region with only minimal formation of the condylarcartilage (Fig. 4, C and D, and figs. S6 and S8). By 6 months, theossification progressed and the articular cartilage formed at the con-dylar surfaces (Fig. 4D and figs. S6 and S8). Only small amounts of theoriginal scaffold material were present within the newly formed con-dyle. By contrast, reconstruction with tissue-engineered bone resultedin the most complete condyle regeneration (Fig. 4, C and D, and figs.S6 and S9). New bone regenerated at the rostral side and from thecaudal end into the tissue-engineered bone graft. Sites of endo-chondral ossification could be observed at the condylar surfaces,within the implanted tissue-engineered bone grafts (Fig. 4 and fig.S10). After 6 months, the engineered bone graft was mostly replacedby the newly regenerated condyle containing a continuous layer ofcondylar cartilage (Fig. 4D and figs. S6 and S9). The presence ofportions of the implanted tissue-engineered bone indicated continuedregeneration and remodeling.

Graft-host integrationThe structural integration of the implanted graft with native bone wasinvestigated using high-resolution mCT and histological analysis.Paired histological (Movat’s pentachrome stain) and mCT image anal-ysis distinguished the implanted scaffold material (both acellular andcell-laden) from the native bone (Fig. 5A). Among the three experi-mental groups, the tissue-engineered grafts most strongly enhancedthe graft-host bone integration. 3D reconstruction of engineeredgraft-host interface displayed the progression of structural integrationbetween the new bone and the host bone at 3 months. Integration wasconfirmed by histology showing new bone penetrating into the graft(Fig. 5A). Higher-magnification histology revealed the proximity ofthe new bone and the resorbing scaffold, as well as the presence of

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osteoclastic processes involved in the remodeling and replacement ofthe graft material with the new bone. By 6 months, a large portion ofthe tissue-engineered bone graft was replaced by new bone (Figs. 4, Band D, and 5A). In contrast, the implanted acellular scaffolds resultedin disconnected mineral structures with soft fibrous tissue formed be-tween the host and the graft.

The mechanical integrity of regenerated bone was analyzed bythree-point bending (fig. S11) to determine structural stability of re-generating bone. The peak and equilibrium flexural moduli of thewhole ramus were significantly higher for the tissue-engineered bonegroup than the acellular scaffold group at both 3- and 6-month timepoints but were still twofold lower than the corresponding values innative bone (Fig. 5, B and C). However, the peak force during bendingof the ramus after engineered bone implantation was similar to that ofthe native bone, indicating that the whole ramus tissue can withstandthe same amount of flexural load as the native bone (Fig. 5C).

The mechanism of graft-host integration was investigated byevaluating the amounts and distributions of type I collagen andBSP. Preosteoblastic cells secreted rich ECM that underwent miner-

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alization to form new bone containing osteocytes in the ramusregion (Fig. 6). The new bone matrix was rich in type I collagenand BSP and ossified (Fig. 6). The cellularized region of this bone-likematrix was present before implantation (Fig. 2C) and maintainedthroughout the implantation study, suggesting that the osteogenicallyinduced ASCs facilitated new bone formation and enhanced graft-hostbone integration.

Scaffold structure, graft resorption, and vascular infiltrationQuantitative analysis of tissue microstructure was used to investigatethe effects of stem cell incorporation on graft structure, resorption,and vascular infiltration. Large portions of the original graft materialwere still present 3 months after implantation (Fig. 7A), with thelargest amount of the original scaffold material present in the tissue-engineered bone graft, as determined by quantitative image analysis(Fig. 7, A and B), presumably because of lower rate of resorption (Fig.7C). In contrast, the acellular scaffold underwent massive resorption invivo with small dense macrophage-like cells infiltrating the grafts andamorphous areas consistent with tissue necrosis (Fig. 7A).

Fig. 3. Progression of bone regeneration and reestablishment of tional images are in fig. S2B. (B to D) Quantitative analysis of bone vol-

condyle height. (A) Lateral (left) and caudal (right) 3D views of themandible after surgery. Images were taken at timed intervals for condy-lectomy controls (n = 1), animals implanted with acellular scaffolds (n =2), and animals implanted with tissue-engineered bone (n = 2). Addi-

ume (B), bone volume fraction (C), and condyle height normalized tothe original height (D). Data are individual animals with means ± SD(n = 6). ***P < 0.001; **P < 0.01; *P < 0.05, two-way ANOVA and Bonfer-roni post hoc tests.

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Notably, the tissue-engineered grafts vascularized, and the newlyformed vasculature was continuous with the host circulation, as evi-denced by the presence of blood cells inside lumen structures inMovat’s pentachrome stains (Fig. 7A). Staining with hematoxylinand eosin (H&E) showed the formation of new vasculature, andthe Prussian blue stain confirmed the presence of hemoglobin (iron)in the cells found within the new blood vessels (Fig. 7D). The endo-thelial lining of the new blood vessels was confirmed by CD31 staining.Vasculature was also present in the acellular scaffold implantation, butto a markedly lower extent than in the tissue-engineered bone grafts

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(Fig. 7, A and E). The vascularization was detected throughout thetissue-engineered bone grafts (Fig. 4, B and D, and fig. S9), indicatingthat the formation of new bone and vascular perfusion occurred inparallel.

DISCUSSION

We developed a translational approach for growing anatomically correctbone grafts from ASCs and demonstrated its utility for reconstruction

Fig. 4. Morphology and structure of regenerated RCU. (A and B) Ramusregeneration was assessed by mCT (A) and Movat’s pentachrome staining

magnification (bottom; 2-mm scale) of the condylectomy site. The dashedcircumferences indicate the remaining graft regions, with the red trabecular

for bone and soft tissues (B). Scale bars, 1 cm. (C and D) Condyle regenera-tion was assessed using mCT 3D reconstruction (C) and Movat’s penta-chrome staining (D) at low magnification (top; 1-cm scale) and high

structure representing the remaining scaffold material. Images are repre-sentative of n = 2 (condylectomy control), n = 4 (acellular scaffold), andn = 4 (tissue-engineered bone) animals.

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of the entire facial bone in a skeletally mature large animal. Cells wereinfused into an anatomically shaped decellularized bone scaffold andcultured in a matching bioreactor chamber with interstitial flow ofculture medium to engineer customized bone grafts. For each animal,the scaffold and the bioreactor were designed by image-guided fabri-cation. Over 6 months after implantation, the engineered grafts rees-tablished the original RCU, integrated with the host bone, and formedbone-like tissue that was extensively vascularized, in contrast to bothgroups of control animals. This study shows successful tissue-engineeredreconstruction of large, anatomically precise, load-bearing cranio-facial defect.

Previously, the utility of osteogenic stem cells has been studied insmall-animal models for craniofacial bone reconstruction (7, 8) and insheep for regeneration of long bones by using MSCs seeded into scaf-folds containing BMP-2 protein (10). We investigated regeneration offlat bones in the skull that involves intramembranous ossification pre-ceded by callus formation (22, 23). Our system did not require addi-tion of BMP-2, as the scaffold alone had strong osteogenic properties.

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For clinical impact, we created anatomically precise complex bonegrafts using decellularized bone scaffolds and autologous MSCs anddemonstrated their utility for reconstructing load-bearing maxillofacialbone defects in a large-animal model.

RCU, the most eminent load-bearing joint of the skull, served as arigorous model for assessing tissue-engineered bone incorporationin skeletally mature minipigs. To mimic the envisioned logistics ofthe commercial process, the grafts were grown and implanted attwo locations that were more than 1200 miles apart. For eachanimal, the CT images and adipose tissues were shipped to the pro-cessing laboratory. Cell preparation and assurance of qualityfollowed good manufacturing guidelines (24–26) so that the processcan be readily adapted to the regulatory requirements. The bio-reactor provided graft cultivation and transport to the implantationsite in a closed system and with the preservation of cell viability. Graftswere implanted by an expert maxillofacial surgeon by followingstandard clinical protocols and using standard plates and screws forfixation.

Fig. 5. Graft-host bone integration. (A) mCT 3D reconstruction of the the lining of osteoblasts (black arrowheads), indicating active ossification.

graft-host interface and Movat’s pentachrome staining were used to assessintegration of the implanted graft with the host bone. For acellular grafts,the mineralized host bone (hb) and the graft structures (g) were separatedby soft fibrous tissue (f). In contrast, host bone extended into the tissue-engineered bone graft. In the proximity of the new bone, osteoclastic re-sorption (white arrowheads) was detected on the implanted scaffold with

Scale bars, 1 mm (4×) and 100 mm (40×). (B and C) Three-point bendingmechanical test for peak and equilibrium flexural moduli (B) and the peakand equilibrium forces (C). Data are means ± SD (n = 4 from two animalsfor condylectomy; n = 4 from two animals for 3-month data; n = 8 fromfour animals for 6-month data). ***P < 0.001; **P < 0.01; *P < 0.05, two-wayANOVA and Bonferroni post hoc tests.

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The study had two controls: condy-lectomy to account for any spontaneoushealing and biomaterial scaffold to ac-count for the role of cells in inducingtissue regeneration. As in previous studiesin large animals (27, 28), condylectomyresulted in minimal bone regeneration.The implanted acellular scaffolds re-sulted in formation of fibrous tissue, ap-parently due to insufficient capacity forreplacing the degrading scaffold by newbone tissue. Only the tissue-engineeredbone grafts enhanced condyle regenera-tion to the original height at both 3 and6 months while maintaining mechanicalintegrity of the ramus. The condylar car-tilage formed at the articular surface andthe subchondral bone replaced the im-planted bone graft, resulting in high fi-delity of the regenerated condylarsurface. Unlike acellular scaffolds, thescaffold material in tissue-engineeredbone grafts was degraded slowly andreplaced by new bone. Fast resorption ofacellular scaffolds caused inflammatory re-sponses and soft tissue infiltration thatpersisted through 6 months of implanta-tion. These properties of acellular scaffoldshave led to granulation and fibrosis atthe graft-host interface, precluding graftincorporation (29). In contrast, engineeredbone grafts enhanced bone ingrowth,provided mechanical stability for mas-tication, and accelerated graft turnoverand new bone formation. These proper-

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ties are important for clinical translation as the success in clinical bonegrafting depends on graft resorption, bone incorporation, and me-chanical stability. Clearly, graft resorption needs to be balanced withthe deposition and remodeling of the new bone for adequate cranio-facial reconstruction (30). We believe that slower bulk resorption andthe presence of osteoclasts in tissue-engineered bone supported favor-able remodeling into native-like bone.

Similar to previous studies that used perfusion bioreactors forbone tissue engineering (16, 21, 31), the grafts contained large num-bers of cells (≥6 × 108 cells) expressing osteogenic markers CBFA1and Osterix (32, 33) with dense bone ECM filling the scaffold pores.The differentiating ASCs expressed high levels of bone-related genesand proteins, such as ALP, type I collagen, BSP, and osteonectin.Notably, BMP-2 [commonly added to support bone culture and repair(34)] and transforming growth factor–b2 (35, 36) were also highlyexpressed in engineered grafts at 3 and 6months after implantation. Con-sistent with previously shown immunoprotective and anti-inflammatoryproperties of MSCs, including ASCs (37–40), tissue-engineered bonegrafts did not express proinflammatory genes.

Vascularization of implanted tissues has been a major challenge, asinadequate nutrient transport leads to necrosis (41). Culture-expandedstromal/stem cells have been shown to promote endothelial migrationand induced angiogenesis both in vitro and in vivo (42–44). Here, vas-

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cularization was associated with the formation of new bone in the re-generating RCU and detected deep inside the graft, as in autologoustrabecular bone implants, where bone was vascularized rapidlythrough the marrow spaces (2). These regenerative features werenot seen in acellular scaffolds, suggesting that the autologous ASCsexhibited osteoconductive and osteoinductive properties as reportedfor autologous trabecular bone implants (2). Nevertheless, the com-parison between engineered bone grafts and tissue autografts needsa more detailed evaluation of the integrative repair at the graft-hostinterface and vascular perfusion.

We demonstrated that bone grafts engineered to the exact anatom-ical shape using decellularized bone scaffolds and autologous ASCsregenerated the entire RCU in a pig model. However, our study alsohas limitations. We did not determine whether enhanced bone forma-tionwas a direct effect of implanted cells or a result of secondary factors,such as immunemodulation, proangiogenic signaling, and/or release ofchemotactic or homing signals. Cell lineage tracking will be required todiscern possiblemechanisms. It will also be of interest to compare ASCsto alternative sources of autogenous cell populations and to determineeffects of cell concentration and the duration of bioreactor culture onintegrative repair. Last, translation of tissue-engineered grafts into clin-ical use will require characterization of ASCs from diverse donor popu-lations to ensure quality control and safety.

Fig. 6. Deposition of type I collagen and BSP in the newly formed bone. The regenerating newbone (nb) within the tissue-engineered bone graft is shown at low and high magnification. Localized

areas of type I collagen and BSP at the graft-host bone interface were precursors for bone formation.New bone contained osteocytes (black arrowheads) within their lacunae. Negative immunohisto-chemistry confirmed the specificity of the staining. Images are representative of n = 4 animals. Scalebars, 400 mm (low) and 100 mm (high).

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MATERIALS AND METHODS

Study designThe objective of our study was to engineer anatomically correct livingcraniofacial bones by using ASCs without BMPs, native bone matrix, aperfusion bioreactor, and image-guided fabrication methods. Our pre-specified hypothesis was that the immature bone formed in vitro willserve as a template for bone development and remodeling and itsfunctional integration with adjacent host tissues. The research was

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conducted using skeletally mature Yu-catán minipigs [21 to 32 months old;48 to 73 kg (60.0 ± 7.9 kg)]. Samplesize was determined using poweranalysis (b = 0.1 and a = 0.05) withmA and mB of 0.4 and 0.6, respectively,and SD of 0.1 based on estimatedbone volume fraction. The resultingsample size was 6.

Anatomically correct engineeredbone grafts were investigated fortheir capacity to regenerate the en-tire RCU during a 6-month periodof implantation in Yucatán mini-pigs. Two control groups were stud-ied: condylectomy to assess anyspontaneous regeneration of thebone and acellular, anatomicallyshaped scaffolds to assess the roleof exogenous cells in tissue regenera-tion. Tissue outcomes were evaluatedby using CT and mCT imaging, geneexpression, protein contents, histolo-gy, immunostains, and biomechanicaltesting. Selection of animals for themain experimental group and thetwo control groups was randomized.In the main experimental group, thecells were autologous and thereforederived from each animal. Also, thescaffold and bioreactor chamber werespecifically made for each animal to fitthe defect geometry by image-guidedfabrication. The acellular scaffoldgroup also required the image-guidedpreparation of a specific scaffold foreach animal. The implantations couldnot be blinded because of the differentappearance of the engineered bone(tissue-like) and acellular scaffold(biomaterial-like). Nevertheless, allanalytical assessments were blinded tothemaximumpractical extent, andmostof them were done by an independentexpert (such as mCT, CT, histology,and mechanical testing) to eliminateany bias.

Stromal/stem cell preparationSubcutaneous fat (about 20 g) was harvested from the dorsal lumbarregion of each animal (n = 7) at the same time as the initial CTprocedure and the adipose-derived stemcells were isolated as previouslydescribed (25). The flasks containing passage 0 (P0) ASCs were com-pletely filled with culturemedium and shipped on ice to ColumbiaUni-versity for processing. The pASCs were expanded in Dulbecco’smodified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS),

Fig. 7. Graft resorption and vascularization. (A) Movat’s pentachrome stain of the central region with-in the scaffolds is shown 3 months after implantation. Scaffold resorption is indicated by white arrows.

Vasculature is indicated by black arrows. Scale bars, 1 mm and 250 mm. (B and C) Quantification of mi-crostructure properties: percentage of scaffold area (B) and percentage of the resorbing area (C). (D) H&Eshowed structural differences between the newly formed vasculature and the surrounding tissue. Immu-nohistochemistry for CD31 confirmed the presence of endothelial cell lining in newly formed vessels in bothtissue-engineered bone and acellular scaffold group. Negative control confirmed the specificity of CD31antibody. The CD31 immunohistochemistry was used to quantify vascular number per area. Scale bar, 50 mm.(E) The number of blood vessels per area was quantified from CD31 immunohistochemical images. (B, C,and E) Data are box plots for n = 56 images for the acellular scaffold group and n = 90 images for thetissue-engineered bone group.

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1% penicillin/streptomycin, and fibroblast growth factor (0.1 ng/ml),and passaged by trypsinization up to P3.

Scaffold fabricationThe scaffolds were made from native bovine bone after removing allcellular material to leave behind the ECM with largely preservedcomposition, architecture, and mechanical properties. The decellular-ization method has been demonstrated to remove all cellular materialfrom the bone (45). Large cancellous bone blocks were created fromthe distal aspect of adult bovine femurs (>2 years old) after removingthe epiphysis, articular surface, and periosteum with an electrical bandsaw. The blocks were lathed into cylinder blocks that are 4.5 cm indiameter and 12 cm long. To fabricate an anatomical TMJ ramus-condyle cancellous bone block, G-code was generated from the InitialGraphics Exchange Specifications (.iges) files of the CT images foreach pig using the Mastercam software. Using a four-axis computer nu-merical control milling machine (LittleMachineShop), the cylindricalbone blocks were milled into the custom geometry of the RCU foreach pig. The anatomical scaffolds were decellularized by using a mod-ification of a known protocol (16) and are described in SupplementaryMethods.

Perfusion bioreactor design and operationThe perfusion bioreactor used in this study was developed startingfrom our previous bioreactor system that was used to generate ana-tomical pieces of tissue-engineered bone with high cell density (16)and is described in Supplementary Methods.

Cultivation of bone graftsThe anatomically shaped scaffolds were seeded and cultured usingperfusion bioreactors with anatomical chambers fabricated to exactlymatch the geometry of each scaffold, as described above. For scaffoldseeding, the scaffold volumes were determined by the SolidWorkssoftware and were in the range of 11 to 15 cm3. A suspension ofP3 pASCs (12 ml; 107 cells/cm3 in expansion medium) was injectedinto the culture chamber throughout the anatomical scaffold. Afterallowing the cells to attach for 3 hours at 37°C in an incubator, oste-ogenic media [low-glucose DMEM supplemented with 10% FBS, 1%antibiotics, 10 mM sodium b-glycerophosphate, 100 nM dexametha-sone, and ascorbic acid 2-phosphate (50 mg/ml)] were perfusedthrough the scaffold for 3 days at 10% of the calculated optimal flowrate to promote spatially uniform cell attachment throughout the scaf-fold volume.

For cultivation of bone grafts, the seeded scaffolds were perfusedwith osteogenic medium for 3 weeks at the set optimal flow rate, withmedium changes twice per week. The 3-week cultivation period wasselected on the basis of the finding that ASCs highly express osteogenicmarkers after 3 weeks of in vitro differentiation (fig. S2). Four engi-neered constructs were generated for each animal: one for implantationand three for evaluation, after 3 days and 3weeks of cultivation and aftertransport from New York, NY, to Baton Rouge, LA, where the graftswere implanted. Transport of the bone grafts is described in Supple-mentary Methods.

Animal implantation studyThe animal implantation study was conducted at the Louisiana StateUniversity under an approved Institutional Animal Care and UseCommittee protocol. A total of 14 skeletally mature Yucatán minipigs

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were divided into three groups: (i) condylectomy (n = 2), (ii) acellularscaffold implantation (n = 6), and (iii) autologous engineered boneimplantation (n = 6). Two surgeries were performed per day over aperiod of 2 months. Two pigs per group receiving acellular scaffold orautologous tissue-engineered bone graft were sacrificed at 3 monthsafter implantation. The remaining animals were sacrificed at 6 months.CT imaging was performed immediately and at 6 weeks, 3 months,and 6 months after surgery. End point analyses included mechanicaltesting and histological and immunohistological evaluation.

All pigs underwent clinical CT scan (GE LightSpeed 16; 120 kilovoltpeaks, 625-mm resolution) 2 to 3 months before surgery. For eachanimal, the facial skull structures were reconstructed from CT imagesusing the Mimics software. From the reconstructed 3D images, weselected the left TMJ RCU measuring 3 cm along the dorsal planeby 6 cm along the transverse plane as the region for defect creationand reconstruction. For all animals except the condylectomy con-trols, the 3D structure data were sent to the laboratory at ColumbiaUniversity and the defect/grafting region was selected in Mimicssoftware.

The animal implantation process and the selection of fixativemethod were chosen by an expert maxillofacial surgeon to closelymimic the current clinical procedure. The implantation procedureoccurred after 12 hours of food withdrawal. The pigs were admin-istered intramuscularly with ketamine (10mg/kg; Vedco Inc.), midazolam(0.2 mg/kg; Hospira Inc.), and dexmedetomidine (2 mg/kg; PfizerAnimal Health). Anesthesia was induced 15 min later with 5% iso-flurane in 100% oxygen at flow (1.5 liters/min) via facial mask. Theanimals were intubated with a cuffed Murphy’s endotracheal tube (7 to9 mm internal diameter), and anesthesia was maintained at a vaporizersetting of 1.5% isoflurane in a circular breathing system.

The pigs were then prepped and draped in a standard sterile fash-ion. We used a retromandibular approach with 15 blades to make a5-cm skin incision. We then used electrocautery to dissect through thesubcutaneous and superficial cervical fascial layers. Blunt dissectionwas carried to the angle and inferior border of the mandible, wherethe periosteum was approached. The facial vessels were identified andligated. The periosteum was incised with electrocautery, and sub-periosteal dissection exposed the medial and lateral mandible fromthe antegonial notch to the head of the condyle. Preplanned cuts weremeasured and marked, and condylectomies were performed with areciprocating saw under copious sterile saline irrigation. The RCUwas then removed and the pig was treated with either no replacement(condylectomy) or replacement with an acellular scaffold or a tissue-engineered bone graft. Two eight-hole miniplates with 1.7-mm bonescrews (Stryker) were used to provide rigid fixation of the implants.The wounds were closed with 3-0 polyglactin 910 sutures for sub-cutaneous tissues and 2-0 nylon sutures for skin.

CT and mCT imagingThe animals were anesthetized and placed in ventral recumbency onthe GE LightSpeed 16 Slice CT scanner (GE Medical Systems). Thescans (0.625-mm resolution; 120 kV) were conducted on all animalsat 2 months before surgery, immediately, and at 6 weeks, 3 months, and6 months after surgery. mCT imaging (50-mm resolution; 100 kV) ofharvested RCUs was done using the GE Explore CT-120 (GE MedicalSystems) at an external facility (Cornell University Imaging Facility) ina blinded fashion. Condyle height and bone volume were measuredusing the Mimics software (Materialise). Quantitation of bone volume

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and bone volume fraction was conducted by determining the amountof bone in the defect area on the basis of the difference between theCT before and after surgery and the amount of bone removed.

mCT was performed by using a modified protocol (45), and thesamples were scanned at 21-mm isotropic resolution. The bone volumewas obtained from the application of a global thresholding techniqueso that only the mineralized tissue was detected. There was no diffi-culty in distinguishing the grafted material from the miniplates andscrews used for fixation, which appeared in mCT as compact and ge-ometrically defined structures. Spatial resolution of the full voxelmodel was sufficient for evaluating the microarchitecture of the bonetissue.

Mechanical testingA simple three-point bending mechanical test was used to assess struc-tural rigidity of the ramus (fig. S11). Samples were cut along dorsalplane into about 1 cm wide (ventral-dorsal) and 3 cm long (cranial-caudal). Two 1-cm sections were cut and tested for each animal sacri-ficed at 3 months and for condylectomy group, and one 1-cm sectionwas cut and tested for other animals. Soft tissue was removed to ex-pose the hard tissue structure. The samples were placed on twosupports that were 25 mm apart. An actuator applied a load in theexact middle of the two supports onto the samples. Preload of 25 gwas applied for 900 s before the test. The samples were subjected to a300-mm displacement at a loading rate of 3 mm/s and then held inplace for 1800 s. Force was measured throughout the period ofloading. Peak force and equilibrium force were determined as themaximum force and the force at the end point of the testing. The flex-ural modulus (E) was calculated from the force measured and the ge-ometry of the sample:

E ¼ Fl3

48yI

where F is force, l is the distance between two supports, y is thedisplacement, and I is the overall second moment of area, whichwas calculated using a solid rectangular beam model:

I ¼ wh3

12

where w and h are width and height of the samples, respectively.

Statistical analysisStatistical analysis was conducted using GraphPad Prism software(GraphPad Software Inc.). Statistical analysis of graft cellularity wascarried out by one-way ANOVA nonparametric test and Tukey’scomparison posttest to compare means. Statistical analysis of gene ex-pression was carried out by the one-sample t test with hypotheticalvalue of 1. Statistical analyses of the bone volume, bone volume frac-tion, condyle height, and mechanical properties were carried out bytwo-way ANOVA and Bonferroni post hoc tests to compare themeans. Data were calculated as the means ± SD. Statistical analysesof image quantitation were carried out by Student’s t test. P < 0.05was considered significant. The box plots were constructed to representmedian, first quartile, and third quartile, with error bars indicatingmax-imum and minimum values; if outliers are present, they are shown asdots above or below. Histograms are means ± 1 SD. Dot plots are indi-vidual animals with lines to represent the means ± 1 SD.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/343/343ra83/DC1MethodsFig. S1. Structural comparison of tissue-engineered graft and native explant.Fig. S2. Regeneration of RCU.Fig. S3. High-resolution Movat’s pentachrome stain of the ramus in the condylectomy group at 6months.Fig. S4. High-resolution Movat’s pentachrome stain of the ramus in the acellular scaffold group.Fig. S5. High-resolution Movat’s pentachrome stain of the ramus in the tissue-engineered bonegroup.Fig. S6. Condyle regeneration (posterior view and histologies).Fig. S7. High-resolution Movat’s pentachrome stain of the condyle in the condylectomy groupat 6 months.Fig. S8. High-resolution Movat’s pentachrome stain of the ramus in the acellular scaffold group.Fig. S9. High-resolution Movat’s pentachrome stain of the ramus in the tissue-engineered bonegroup.Fig. S10. Ossification patterns in the condyle after tissue-engineered bone regeneration.Fig. S11. Experimental setup for the three-point bending mechanical testing.Table S1. Porcine real-time polymerase chain reaction (RT-PCR) primers.Movie S1. Assembly of bioreactor.Movie S2. Finite element modeling of oxygen concentration.

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Acknowledgments: We thank the staff at the Louisiana State University School of VeterinaryStudy for veterinary assistance. Funding: We gratefully acknowledge funding support by NIH(grants DE016525 and EB002520), the New York Partnership Bioaccelerate Program (CU11-1915), and Mikati Foundation (gift funding for research). Author contributions: S.B., S.B.E.,and G.V.-N. designed experiments and developed the animal model. S.B. and G.V.-N. wrotethe paper. S.B., K.Y., and R.E.E. were responsible for bioreactor design and fabrication. F.S.and J.M.G. isolated and characterized ASCs. S.B., J.C.B., and R.E.E. were responsible fortissue-engineered bone graft manufacturing, studies of the osteogenic differentiation,mechanical testing, histology, immunohistochemistry, CT, and mCT analysis. J.C.B. performedRT-PCR analysis and blinded image quantitation. R.E.E. performed DNA quantitation. D.M.A., M.J.L.,and S.B.E. performed animal surgery. J.B. and M.J.L. harvested adipose tissue and oversaw animalcare. Competing interests: The perfusion bioreactor technology described in this paper is patent-pending (WO 2014172575 A1) and is now being commercialized through a Columbia Universityspinout, EpiBone, cofounded by S.B., S.B.E., and G.V.-N. J.M.G. is a co-owner, cofounder, and chiefscientific officer of LaCell LLC, a for-profit company focusing on stromal/stem cell technology andclinical translation. Data and materials availability: All materials are available from commercialsources or can be derived using methods described in this study. All relevant data are reported inthe article and the Supplementary Materials.

Submitted 6 October 2015Accepted 27 April 2016Published 15 June 201610.1126/scitranslmed.aad5904

Citation: S. Bhumiratana, J. C. Bernhard, D. M. Alfi, K. Yeager, R. E. Eton, J. Bova, F. Shah,J. M. Gimble, M. J. Lopez, S. B. Eisig, G. Vunjak-Novakovic, Tissue-engineered autologousgrafts for facial bone reconstruction. Sci. Transl. Med. 8, 343ra83 (2016).

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Tissue-engineered autologous grafts for facial bone reconstruction

Jeffrey M. Gimble, Mandi J. Lopez, Sidney B. Eisig and Gordana Vunjak-NovakovicSarindr Bhumiratana, Jonathan C. Bernhard, David M. Alfi, Keith Yeager, Ryan E. Eton, Jonathan Bova, Forum Shah,

DOI: 10.1126/scitranslmed.aad5904, 343ra83343ra83.8Sci Transl Med

with craniofacial bone deformities.anatomically correct, large-scale bone constructs could vastly improve regenerative medicine options for patientstissue, formed new bone, and was vascularized extensively, but only if cells were present. Growing such surgery. Paired histological and image analysis showed that the implanted scaffold material integrated with hostreconstructing human facial bones, then authors shipped the bioreactor with the living bone inside the site of cultured on the bone for several weeks. To mimic the manufacturing and transport chain that could be involved inminipigs, which have similar jaw anatomies and weight-bearing properties as humans. Then, stem cells were a custom-designed perfusion bioreactor. The bone was first shaped to the defect in the ramus-condyle unit ofdesigned a maxillofacial reconstructive strategy centered on a combination of stem cells, decellularized bone, and by providing personalized grafts for deformities of all shapes and sizes. Bhumiratana and colleagues thereforecurrently uses bone grafts from the same patient. Cell- and biomaterial-based approaches could benefit this field

reconstructionsaving face through restoration of actual bone structure. A delicate and precise process, facial bone Restoring your reputation after a social gaffe may be challenging, but perhaps welcomed in comparison to

Saving face

ARTICLE TOOLS http://stm.sciencemag.org/content/8/343/343ra83

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2016/06/13/8.343.343ra83.DC1

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http://stm.sciencemag.org/content/scitransmed/12/534/eaay6853.fullhttp://stm.sciencemag.org/content/scitransmed/10/453/eaao6806.fullhttp://stm.sciencemag.org/content/scitransmed/10/446/eaaq1802.fullhttp://stm.sciencemag.org/content/scitransmed/5/191/191ra83.fullhttp://stm.sciencemag.org/content/scitransmed/3/68/68ra9.fullhttp://stm.sciencemag.org/content/scitransmed/4/141/141ra93.fullhttp://stm.sciencemag.org/content/scitransmed/4/160/160rv12.full

REFERENCES

http://stm.sciencemag.org/content/8/343/343ra83#BIBLThis article cites 45 articles, 7 of which you can access for free

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