15
Bone Tissue Engineering Bioreactors: A Role in the Clinic? Erin Salter, B.S., Brian Goh, B.S., Ben Hung, B.S., Daphne Hutton, B.S., Nalinkanth Ghone, Ph.D., and Warren L. Grayson, Ph.D. Tissue engineered bone grafts have the potential to be used to treat large bone defects due to congenital abnormalities, cancer resections, or traumatic incidents. Recent studies have shown that perfusion bioreactors can be used to generate grafts of clinically relevant sizes and shapes. Despite these scientific and technological successes, there is uncertainty regarding the translational utility of bioreactor-based approaches due to the perceived high costs associated with these procedures. In fact, experiences over the past two decades have demonstrated that the widespread application of cell-based therapies is heavily dependent on the commercial viability. In this article, we directly address the question of whether bioreactors used to create bone grafts have the potential to be implemented in clinical approaches to bone repair and regeneration. We provide a brief review of tissue engineering approaches to bone repair, clinical trials that have employed cell-based methods, and advances in bioreactor technologies over the past two decades. These analyses are combined to provide a perspective on what is missing from the scientific literature that would enable an objective baseline for weighing the benefit of extended in vitro cultivation of cells into functional bone grafts against the cost of additional cultivation. In our estimation, the cost of bioreactor-based bone grafts may range from $10,000 to $15,000, placing it within the range of other widely used cell-based therapies. Therefore, in situations where a clear advantage can be established for engineered grafts comprising patient-specific, autologous cells, engineered bone grafts may be a clinically feasible option. Introduction B one grafts are widely used therapeutically for the treatment of large defects arising from cancer resections, congenital diseases, or trauma. More than half a million bone graft surgeries take place annually in the United States with an associated economic burden of $2.5 billion. 1 Tissue engineering approaches have the potential to significantly impact current therapeutic modalities by providing a virtually unlimited supply of bone grafts in anatomical geometries comprised of patient-specific, autologous stem cells. Despite the considerable promise and the purported advantage that they present in terms of providing expedited healing, engineered bone grafts grown entirely in vitro have yet to be utilized clinically to surgically repair a bone defect in a human. In contrast, cell- and scaffold-based approaches have been used in limited clinical settings demonstrating their feasibility. However, engineering bone grafts of clinically relevant sizes requires extended in vitro cultivation of constructs in bioreactors (Fig. 1), and may sig- nificantly increase the cost of the graft. The cultivation of osteoblasts in three-dimensional scaffolds was first reported circa 1990. 2–6 These early experiments were conducted in traditional culture dishes using ‘‘static’’ medium conditions. Static cultures are insufficient for growing cells in large constructs of clinically relevant sizes (centimeters) as the limited diffusion of oxygen and nutrients will support cell viability only through a depth of several hundred microme- ters. 7,8 This problem has been reconciled through the use of perfusion bioreactors, which provide convective mass trans- port of oxygen and metabolites to cells and facilitate the uni- form development of tissue throughout the constructs. 9,10 In addition to greatly enhancing mass transport, perfusion also introduces shear stress stimulation, which enhances osteo- genic differentiation and calcium deposition by bone marrow- derived mesenchymal stem cells (MSCs). 10,11 As a result, bioreactors have been readily adopted by the bone tissue en- gineering field as an enabling technology, capable of playing a crucial role in translating engineered bone grafts into clinically applicable products. In spite of the in vitro successes, there still remains a dis- connect between the technological advances in bioreactor de- velopment and their clinical use. Why does this disconnect exist and what can we do to bridge this gap? The majority of concerns regard cost, time, and ease of application. How practical is it to apply bioreactor-grown grafts in the clinic? Current methods rely heavily on careful manual techniques, which are subject to operator-dependent variability, and make regulatory approval exceedingly difficult to obtain. The use of advanced technology would also require training of special- ized technicians to monitor graft cultivation. Thus, despite its potential scientific utility, many investigators appear to be moving away from this approach to more ‘‘practical’’ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland. TISSUE ENGINEERING: Part B Volume 18, Number 1, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.teb.2011.0209 62

Bone Tissue Engineering in Clinic

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Page 1: Bone Tissue Engineering in Clinic

Bone Tissue Engineering Bioreactors: A Role in the Clinic?

Erin Salter, B.S., Brian Goh, B.S., Ben Hung, B.S., Daphne Hutton, B.S.,Nalinkanth Ghone, Ph.D., and Warren L. Grayson, Ph.D.

Tissue engineered bone grafts have the potential to be used to treat large bone defects due to congenitalabnormalities, cancer resections, or traumatic incidents. Recent studies have shown that perfusion bioreactorscan be used to generate grafts of clinically relevant sizes and shapes. Despite these scientific and technologicalsuccesses, there is uncertainty regarding the translational utility of bioreactor-based approaches due to theperceived high costs associated with these procedures. In fact, experiences over the past two decades havedemonstrated that the widespread application of cell-based therapies is heavily dependent on the commercialviability. In this article, we directly address the question of whether bioreactors used to create bone grafts havethe potential to be implemented in clinical approaches to bone repair and regeneration. We provide a briefreview of tissue engineering approaches to bone repair, clinical trials that have employed cell-based methods,and advances in bioreactor technologies over the past two decades. These analyses are combined to provide aperspective on what is missing from the scientific literature that would enable an objective baseline for weighingthe benefit of extended in vitro cultivation of cells into functional bone grafts against the cost of additionalcultivation. In our estimation, the cost of bioreactor-based bone grafts may range from $10,000 to $15,000,placing it within the range of other widely used cell-based therapies. Therefore, in situations where a clearadvantage can be established for engineered grafts comprising patient-specific, autologous cells, engineeredbone grafts may be a clinically feasible option.

Introduction

Bone grafts are widely used therapeutically for thetreatment of large defects arising from cancer resections,

congenital diseases, or trauma. More than half a million bonegraft surgeries take place annually in the United States with anassociated economic burden of $2.5 billion.1 Tissue engineeringapproaches have the potential to significantly impact currenttherapeutic modalities by providing a virtually unlimitedsupply of bone grafts in anatomical geometries comprised ofpatient-specific, autologous stem cells. Despite the considerablepromise and the purported advantage that they present interms of providing expedited healing, engineered bone graftsgrown entirely in vitro have yet to be utilized clinically tosurgically repair a bone defect in a human. In contrast, cell- andscaffold-based approaches have been used in limited clinicalsettings demonstrating their feasibility. However, engineeringbone grafts of clinically relevant sizes requires extended in vitrocultivation of constructs in bioreactors (Fig. 1), and may sig-nificantly increase the cost of the graft.

The cultivation of osteoblasts in three-dimensional scaffoldswas first reported circa 1990.2–6 These early experiments wereconducted in traditional culture dishes using ‘‘static’’ mediumconditions. Static cultures are insufficient for growing cells inlarge constructs of clinically relevant sizes (centimeters) as thelimited diffusion of oxygen and nutrients will support cell

viability only through a depth of several hundred microme-ters.7,8 This problem has been reconciled through the use ofperfusion bioreactors, which provide convective mass trans-port of oxygen and metabolites to cells and facilitate the uni-form development of tissue throughout the constructs.9,10 Inaddition to greatly enhancing mass transport, perfusion alsointroduces shear stress stimulation, which enhances osteo-genic differentiation and calcium deposition by bone marrow-derived mesenchymal stem cells (MSCs).10,11 As a result,bioreactors have been readily adopted by the bone tissue en-gineering field as an enabling technology, capable of playing acrucial role in translating engineered bone grafts into clinicallyapplicable products.

In spite of the in vitro successes, there still remains a dis-connect between the technological advances in bioreactor de-velopment and their clinical use. Why does this disconnectexist and what can we do to bridge this gap? The majority ofconcerns regard cost, time, and ease of application. Howpractical is it to apply bioreactor-grown grafts in the clinic?Current methods rely heavily on careful manual techniques,which are subject to operator-dependent variability, and makeregulatory approval exceedingly difficult to obtain. The use ofadvanced technology would also require training of special-ized technicians to monitor graft cultivation. Thus, despite itspotential scientific utility, many investigators appear to bemoving away from this approach to more ‘‘practical’’

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland.

TISSUE ENGINEERING: Part BVolume 18, Number 1, 2012ª Mary Ann Liebert, Inc.DOI: 10.1089/ten.teb.2011.0209

62

Page 2: Bone Tissue Engineering in Clinic

alternatives such as growth factor or gene delivery. The futureof bone tissue engineering bioreactors as more than a scientifictool has therefore been called into question. In this review, weexamine the potential for translating this bioreactor-basedapproach to clinical scenarios and discuss key points to beaddressed if we are to overcome the scientific, regulatory, andcommercial barriers that have crippled previous cell-basedtherapeutic approaches and have so far prevented tissue-engineered bone grafts from reaching the clinic.

Bioengineering Approaches—A Casefor Cell-Based Therapies

The term ‘‘bone tissue engineering’’ used in its most generalform may involve either cell-free approaches, cell-based ap-proaches, or combinations of both.12 The cell-free approachconsists of scaffolds or bone graft substitutes, which incorpo-rate osteoconductive or osteoinductive attributes based on thescaffold’s physical and chemical properties, added growthfactors, or DNA oligomers for transfection. The cell-basedapproach consists of the use of either autologous or allogeneiccells capable of forming bone tissue with or without the use ofscaffolds. The performance of these cells for bone regenerationmay be further enhanced by genetic modifications.

Scaffolds

Advances in biomaterials and nanotechnology have rev-olutionized the field of tissue engineering by providingscaffolds of desired physical, chemical, mechanical, and

biodegradable characteristics. Among the scaffolds madefrom natural polymers, collagen, hyaluronic acid, alginate,and chitosan are the most commonly used.13,14 Thoughnatural polymers have desirable biodegradability, they lackthe proper mechanical properties for bone engineering. Themechanical qualities of natural polymers can be enhanced byblending them with Food and Drug Administration (FDA)-approved synthetic polymers such as polycaprolactone(PCL), polylactic acid (PLA), polyglycolic acid (PGA),poly(lactic-co-glycolic) acid (PLGA), or ceramics such ashydroxyapatite and b- tricalcium phosphate (b-TCP).15,16

Another class of scaffolds that has been extensively studiedand found to be promising for bone tissue engineering ap-plications is synthetic bioactive glasses and demineralizedbone matrix.17,18 The choices are many, each material hasspecific advantages and disadvantages, and the use of hybridmaterials to minimize the disadvantages is an area of in-tensive research.

Bioactive growth factors

These enhance cell growth and differentiation, motility,and formation of new tissues. Key osteoinductive cytokinesinclude bone morphogenic proteins (BMPs)19 and vascularendothelial growth factor (VEGF).20 Among the BMPs, BMP2and BMP7 have been approved for clinical use for thetreatment of nonunion fractures and spinal fusion.21,22 Cer-tain protein mixtures derived from biological source such asblood or tissue, e.g., platelet-rich plasma (PRP), have alsobeen found to be useful for bone tissue engineering.23 Theseagents can be injected directly into bone defect regions orformulated with natural or synthetic polymers for controlledtime release. Because of their short half-lives in vivo, highinitial dosages are required, which in turn can lead to non-specific effects of the growth factors outside of the targetarea. Moreover, the cost associated with the production ofrecombinant proteins is very high. Thus gene therapy hasbeen proposed as an alternative to ensure stable and consis-tent production of osteoinductive proteins at the injury site.24

Gene Therapy

Introducing osteogenic genes to cells in order to upregu-late the expression of bone specific proteins can enhance celldifferentiation and new bone growth. The gene deliveryvector can be carried in a scaffold to induce the local hostcells (in vivo gene therapy) or cells can be genetically modi-fied in vitro and then used for transplantation (ex vivo genetherapy).25 Nonviral gene delivery vectors are relatively safeand cost effective. However, induced gene expression istransient and transfection efficiency is low in comparisonwith viral gene delivery.26 Viral delivery of genes encodingBMPs 2, 4, 6, and 7 has been evaluated for several bonedefects in animal models.27–29 Though the in vivo gene de-livery method appears promising, there is some associateddifficulty in targeting specific cells and innate risk of induc-ing immune responses to the viral vector. For this reason, exvivo gene therapy can be used as an alternative, in which thecells are isolated from the host and genetically modifiedunder controlled in vitro cultures and then transplanted tothe injury site using a cell delivery vehicle. Promising resultshave been observed using ex vivo gene therapy in severalanimal model studies.30,31 However, the effort and time

FIG. 1. Cell-based therapy options for bone regeneration.Current technologies and small-scale clinical trials have uti-lized either direct injection of autologous cells (A) or cell-seeded scaffolds implanted immediately without externalcultivation (B). However, tissue-engineered bone grafts em-ploy ex vivo bioreactor cultivation methods to generate anengineered graft prior to reimplantation (C). In this review,we discuss the potential for translating tissue-engineeredbone grafts to clinical applications, evaluating both therelative advantages as well as scientific, regulatory, andcommercial hurdles. Color images available online at www.liebertonline.com/teb

BONE TISSUE ENGINEERING BIOREACTORS 63

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Page 3: Bone Tissue Engineering in Clinic

involved in isolation, ex vivo culture, transfection, and thentransplantation to the injury site make this therapy expen-sive. Currently bone regeneration using gene therapy is notin clinical use; however, active research is in progress.

Cell-based therapy

Cell-based therapy depends entirely upon the activity ofcells to accomplish the regenerative process. Several basicconsiderations have to be taken into account when selectingthe cell source for bone tissue engineering. Considering theimmunogenic response, an autologous cell source is pre-ferred over an allogeneic source; several autologous cellsources have already been evaluated extensively based uponease of availability, expansion capacity, and functional re-generation.32 Though cell therapy based upon the injection ofcell suspensions is simple to perform, there is low localiza-tion of the cells in defect regions due to the possibility ofmultiple homing sites in vivo.33 A combination of encapsu-lating the cells in a scaffold and providing the necessarygrowth factors and microenvironment during ex vivo cul-tures is useful to fully optimize the cells’ regenerative cap-abilities.34

Clinical Applications of Cell-Based Approachesto Treating Bone Defects

The gold standard for autologous bone repair remains thevascularized autograft. However, in cases where this ap-proach is not feasible, notably in patients with donor sitemorbidities such as infections, pain, and hematomas, allo-graft methods have been pursued. Fresh allografts, however,have associated risks of disease transmission, such as HIV,hepatitis B, and hepatitis C. Stem cell-based approaches,where the patient’s own cells are harvested and used to en-hance bone regeneration, have been seen as a viable alter-native. Clinical trials have largely been divided intoprocedures that utilize scaffolds in addition to cells and thosethat are solely cell based. This division is largely dictated bythe size of the bone repair that is needed. Larger defectstypically require a scaffold to primarily fill the void as well asprovide mechanical strength and cellular containment.Smaller defects such as nonunions are oftentimes treatedwith cell suspensions, as there is less of a need for a load-bearing scaffold. Several of these cases have been reportedand extensively reviewed in the literature.35 These ap-proaches and outcomes are summarized in Table 1.

Although there has not been an abundance of clinical trialsof cell-based treatments, there has been promising prelimi-nary data to suggest progress in the field. For example, theapplications of bone marrow (BM) cells to promote boneunionization were shown by Connolly et al. in a seminalstudy.36 In this study, 20 cases of tibial nonunions weretreated with BM aspirate infusion. Of these, half exhibitedcomplete union with intramedullary nailing and 8 out of theremaining 10 demonstrated unionization with cast immobi-lization.36 Subsequent BM studies optimized the conditionsneeded for adequate bone repair. Repeated injections of BMaspirate and increased cell concentrations were both dem-onstrated in separate studies to improve bone unioniza-tion.37–39 With the knowledge that more cells could result inbetter outcomes, subsequent studies proposed expanding theBM aspirate ex vivo and differentiating the cells in osteogenic

medium before implantation.40 This procedure of ex vivoexpansion of the BM aspirate quickly became the preferredmethod of increasing the numbers of osteogenic cells sincethey are very limited in quantity in BM aspirate. The abilityto osteogenically differentiate the BM cells before implanta-tion also resulted in shorter healing time of 30.0 – 6.72 days/cm of defect in comparison to 51.4 – 26.5 days/cm in no celltherapy at all.40

In an effort to standardize preliminary findings, therehave been a few randomized, controlled trials using BM cellsto improve bone defect outcomes. In 2008, Wright et al.performed a study in which they randomized patients toeither BM injections or methylprednisolone therapy in thetreatment of bone cysts. Although there was healing with celltherapy, it was demonstrated that the standard of care,methylprednisolone, had a greater impact on patient out-comes.41 However, several more recent studies have con-tradicted these results, showing that there is tremendoussignificance in the details of the methods utilized for celltreatment. Increased cell number, repeat injection of BM as-pirate into the site of injury, and ex vivo expansion and dif-ferentiation can all increase the capacity of bone marrow cellsto repair a defect.37–40 To ensure the success of cell implan-tation and minimize the amount of marrow that must betaken from the patient, ex vivo expansion and differentiationshould be pursued as an attractive option.

To treat larger bone defects, cells have been combinedwith biocompatible scaffolds to improve form and function.The scaffold provides cellular containment, enhancing de-livery of cells to the affected site, and immediate mechanicalstability while the BM aspirate facilitates bone healing andremodeling. One of the first studies to combine the two totreat defects in humans was in 2001 by Quarto et al., whoused macroporous hydroxyapatite scaffolds seeded with exvivo expanded BM cells.42 Many studies, following thismodel in which hydroxyapatite was used as a cell scaffold,have demonstrated appropriate healing.42–44 In addition,various scaffold materials, including type I collagen andbeta-tricalcium phosphate, have been used with mixed re-sults.45–47 Unfortunately, extensive in vivo studies have notbeen done to directly compare effects of scaffold materials onbone healing properties. The natural progression in BM/scaffold combinations was to include a substance to maintaincells within the scaffold. This was first accomplished byDallari et al., who performed a prospective, randomizedstudy that incorporated platelet gel into lyophilized bonechips. The addition of cell containment by gel strongly in-creased the bone healing and revascularization.45 Despite itssuccess, gel applications have not been widely employed forcell delivery in cell/scaffold-based therapies.

To date, cell-seeded scaffolds have been used in clinicaltreatments without any precultivation to stimulate the or-ganization of cells into immature tissues. It remains largelyunknown whether in vitro cultivation—used to convert cell-seeded constructs into engineered bone grafts—would pro-vide enhanced outcomes. Prior results from small animalstudies suggest that cultured bone grafts lead to improvedhealing of critical sized segmental defects. Meinel et al.demonstrated that BM cell seeded silk scaffolds that weregrown in a bioreactor for 5 weeks before implantation ex-hibited more bone formation as seen by histology and radi-ology than cells alone or scaffolds that were seeded just prior

64 SALTER ET AL.

Page 4: Bone Tissue Engineering in Clinic

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65

Page 5: Bone Tissue Engineering in Clinic

to implantation.48 These results imply that engineered bonegrafts may be particularly suitable for treating critical sizedefects. This is especially relevant in cases where a staticstructural support is insufficient such as congenital defectswhere the graft needs to grow in tandem with a developingchild. To determine whether the additional benefit of grow-ing bone constructs outweighs the costs, head-to-head com-parisons where the long-term outcomes are assessed areneeded in large animal models. These evaluations are miss-ing from the clinical literature. Postoperative radiologicalstudies are not necessarily indicative of functionality. Otherfactors such as fracture reoccurrence and better measures ofclinical utility need to be considered to ensure functional,regenerative success. Also, a better understanding of the rolethat implanted cells play in the healing process is necessaryto confirm that the cell-mediated therapy benefits are in factdue to the cellular component on the implant.

Additionally, as with all clinical approaches to tissue en-gineering, it is of the utmost importance to consider the stemcell source as well as possible donor site morbidity. Ideally,the cell source would have the characteristics of easy acces-sibility, ability to harvest large cell numbers, and an osteo-genic differentiation potential. While BM cells have been thechoice source because of their osteogenic capacity, there arestill complications in their available access and ability toyield high cell counts. Other adult stem cell sources, such asadipose-derived adult stem cells (ASC), have been suggestedas potential primary cell types. ASCs have the advantage ofbeing easily accessible to a large quantity of cells while stillmaintaining osteogenic potential.49 Simple subcutaneous li-posuction can yield approximately 375,000 cells/mL of li-poaspirate after only 4 days of culture.43 Other possible BMstem cell harvesting techniques, such as the reamer-irrigator-aspirator (RIA), have been proposed as prospective methodsto harvest large number of BM stem cells with the addedbenefit of never having to leave the OR.50 RIA also providesa method of obtaining BM stem cells in a putty-like form,which can then be molded to fit the bone defect. These dif-fering cell sources and techniques of harvest are necessaryto consider when approaching clinical applications in tissueengineering as each method has its own benefits and com-plications.

Apart from considerations of cell source and harvestingmethods, it is also important to evaluate the physiologicalstate of the site of implantation as the development and in-tegration of an engineered bone graft postimplantation relyon key osteoconductive conditions. The objective of regen-erative medicine is to not only replace bone deficits but alsohave the graft completely integrate into the recipient patient.To this end, fostering an osteoinductive environment bothlocally and systemically is crucial. For instance, Zhang et al.have explored manipulating bone endocrinology to facilitateregeneration through the application of parathyroid hor-mone in addition to calcium and vitamin D.51,52 These os-teogenic factors ensure that there is ample active calcium forbone repair and integration. Other groups have utilizedcompounds such as bisphosphonates to inhibit osteoclastactivity and prevent bone resorption, shifting the balance topositive bone formation.53,54 Based on this concept of creat-ing the ideal environment to facilitate the integration of im-planted bone constructs, other groups have pioneered theendogenous tissue engineering approach. Enabled by the

idea that the body is the most capable and efficient tissueengineer, it is logical to attempt to harness the body’s innatecapacity for repair. This approach utilizes osteoinductiveproteins or biological agents to stimulate bone repair. Theemergence of BMP supplementation to bone grafts is an aptexample of employing osteogenic agents to manipulate thebody’s own regenerative processes.55 Additionally, othershave experimented with modulating genetic components,such as Lrp5, to regulate bone mass, to achieve better bonerepair.56 However, regardless of the cell origin or harvestingtechniques used, it is this optimization of systemic osteo-conductive conditions that will ultimately determine the re-generative success of engineered bone grafts.

Bone Bioreactors—Growing Functional BoneGrafts in Vitro

Various types of convection-based bioreactors havebeen developed to aid in the cultivation of large, three-dimensional grafts. Goldstein et al.57 compared three suchsystems: the spinner flask,58 the rotating wall vessel,59 andthe perfusion bioreactor,60 to determine the relative effec-tiveness of each. PLGA foam scaffolds were seeded with ratmarrow cells and cultured in the three systems for up to 2weeks. Scaffolds cultured under traditional static conditionswere used as controls. At 7 days, the perfusion system yiel-ded the most uniform cell density throughout the construct,whereas the other systems showed preferential cell density atthe construct boundaries as would be expected for systems inwhich mass transport into the construct center was insuffi-cient for cell viability. By 14 days, only the perfusion systemhad statistically significant levels of alkaline phosphatase(ALP) expression. Together, these results demonstrated thatonly the perfusion system could adequately support uni-form bone differentiation throughout the construct.57

Medium perfusion also imparts shear-stress to the cells.Biophysical forces, such as shear, have been strongly impli-cated in regulating osteocyte function in vivo, and perfusionbioreactors provide a means for mimicking physiologicalloads to 3D cultures of osteogenic cells.61 In 2002, Bancroftet al. seeded rat marrow cells throughout a titanium meshscaffold and showed that matrix and mineral deposition in-creased throughout the scaffold as a function of mediumperfusion rate.61 It was unclear, however, whether this im-provement resulted from enhanced nutrient transport or theincreased biophysical stimulation of cells. The same groupwas able to use their perfusion system and decouple theeffects of shear from that of mass transport by increas-ing medium viscosity while maintaining the same flowrate, demonstrating that the shear applied to cells was amajor factor contributing to de novo bone growth.62 Furtherstudies have investigated specific parameters of the flowthrough tissue engineering constructs to achieve optimalbone formation, such as the effect of oscillatory flow or offlow velocity.10,63 These studies established the ability of theperfusion bioreactor to accommodate tissue engineeringconstructs of clinically relevant sizes. Our group utilized thisapproach while also incorporating grafts with anatomicallyshaped geometry.64 Using a computed tomography (CT)image and computer software to extract the 3D anatomicalshape of the temporomandibular joint condyle, it was pos-sible to generate trabecular bone scaffolds in the exact

66 SALTER ET AL.

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geometry of the condyle. The scaffold, now clinically rele-vant in both size and shape, was seeded with bone marrow-derived mesenchymal stem cells and cultured in perfusionbioreactors for up to 5 weeks. Again, perfused constructsexhibited significantly enhanced matrix formation and min-eral deposition relative to constructs grown in static condi-tions.64 Fluid flow through these constructs was modeledand correlated with patterns of tissue growth and matrixdeposition.64 This represents a critical proof-of-concept thatthe perfusion bioreactor technology can indeed engineerclinically relevant tissues. While a detailed review on theeffects of shear stress and bioreactor culture on cell growth intissue engineered bone grafts is not the focus of this review,two recent reviews cover these topics in depth.65,66

Despite such advances, the perfusion bioreactor has notbeen employed for clinical use. Several reviews of the chal-lenges facing this technology and preventing its widespreaduse have been published.67–69 A critical requirement of atranslational bioreactor is the minimization of operator de-pendency, since this may result in inconsistency and a lack ofquality control between grafts. To facilitate bioreactor use inclinical settings, the user interface must be restricted tonontechnical considerations (e.g., cell injection, mediumchanges, and sampling), as these would enable traditionalhospital staff to operate the system effectively (Fig. 2). It isclear that any such system would need to be equipped withthe following basic features:

1. Automation. Unlike other engineering disciplines inwhich physical and chemical interactions may be ac-curately predicted, accounting for biological complexityand donor-to-donor variations in cellular responses in-troduce challenges for engineering grafts with precise,reproducible qualities. This problem is exacerbatedwhen the quality of the product is heavily dependent onthe individual skill and technique of the operator. Au-tomation of cultivation procedures such as cell seedingreduces variability between manual operators, allowsfor processes to be scaled up to reduce cost, and canlimit the potential for contamination of cultures throughrepeated manual operations. In particular, the ability togrow grafts in duplicate or triplicate for a single ap-plication, and a means to select the ‘‘best’’ graft, mayincrease the chances for successful clinical outcomes.This approach is practical however, only in automatedprocedures enabling process scale-up and the simulta-neous cultivation of multiple grafts. Furthermore, en-hanced quality control is an essential prerequisite forregulatory approval. Advanced technology has alreadyfacilitated automation of a number of clinical processes,for instance, algorithms for precise, image-guided nee-dle placement by surgical robots (termed visual serv-ing) allow for reproducibility in minimally invasivesurgeries.70 Such technology automates the traditionalmanual alignment process surgeons would otherwiseundertake prior to needle insertion. Given our currentcapabilities to model the effects of perfusion rate andmedium flow patterns on tissue development,64 feed-back control might be used to modulate inlet and outletflows in perfusion systems. Likewise, the same princi-ples of automating processes via user-programmedspatial and temporal parameters can also be applied tovarious aspects of bioreactor culture, allowing forminimization of operator-dependent effects on the finalgraft. To achieve this, however, further studies areneeded in the development of relevant quantitativebiomarkers.

2. Uniform cell seeding. The creation of any large graft re-quires uniform cell seeding that results in homoge-neously distributed and viable cells. Bioreactors havelong been employed to this end. In one of the earlieststudies employing spinner flask bioreactors for cellseeding, Vunjak-Novakovic et al. demonstrated uniformcell seeding of poly[glycolic acid] scaffolds up to 10 mmin diameter and 5 mm in height.58 Since then, variousbioreactor designs have been developed to seed scaf-folds. Griffon et al. conducted a study comparing fourdifferent types of bioreactors—the spinner flask, theperfusion system with a vacuum chamber, an orbitalshaker with a vacuum chamber, and an orbital chamberalone.71 This study revealed that seeding with a perfu-sion/vacuum system resulted in *95% cell attachmentwithin the first 2 h and constant cell retention up to 48 hlater. In another study, the tissue culture under perfu-sion (T-CUP) system was developed to simplify thecomplicated set-up required for most perfusion biore-actors.72 In this design, the scaffolds were moved upand down through a ‘‘stationary’’ cell suspension.Using this approach, they were successfully able to seedvarious cell types at high efficiency ( > 75%) and

FIG. 2. Clinical bioreactor for bone tissue engineering. Theprincipal underlying consideration is that the system shouldhave a simple user-interface. The automated bioreactor willhave three user inputs. A porous, prefabricated scaffold isencased within a sterile disposable cartridge. The cartridge isinternally molded to anchor the custom-shaped scaffold andguide perfusion flow evenly through the scaffold. Onceplaced within the bioreactor, input and output perfusionports will pierce the sides of the cartridge. Cells isolated fromthe patient are injected directly into a chamber of the biore-actor pump. The cells are seeded in the scaffold via oscilla-tory, bidirectional perfusion (blue line), followed by a shortperiod of static culture to allow cell attachment. A culturemedium cartridge is connected to the perfusion pump, whichcirculates medium through the scaffold cartridge (red line).An automated sampler (green) will periodically take samplesof the culture medium to assess sterility, pH, oxygen tension,etc. This information will be processed by an external feed-back controller that can adjust the cultivation parameters asnecessary and send alerts to the operator via a graphic userinterface. Once the predetermined cultivation period iscomplete, the engineered graft can be transported in itsoriginal cartridge to the operating room. Color imagesavailable online at www.liebertonline.com/teb

BONE TISSUE ENGINEERING BIOREACTORS 67

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viability ( > 95%) in a variety of scaffolds. These studiesfurther demonstrated the feasibility of utilizing perfu-sion bioreactor system not only in providing masstransport and mechanical stimuli, but also in effectinghomogeneous cell seeding. This is an essential compo-nent of a translatable bioreactor system.

3. Checkpoint markers and feedback control. Relevant bio-markers are needed to serve as checkpoints throughouttissue development, allowing engineers to track thedevelopmental progress of each graft. Yet, to date, norigorous correlations have been established betweenspecific biomolecules and particular stages of bone de-velopment or construct mineralization. Investigationinto bone cytokines has pointed to interleukin (IL)-11 asa possible candidate biomarker. IL-11 is secreted bydeveloping osteoblasts and is crucial for bone re-modeling, meaning it would be present throughout adeveloping bone graft.73 Since IL-11 is a soluble factor,it can be detected noninvasively with a tagged antibodyin the media, or by incorporating available quantifica-tion technologies (such as ELISA). However, furtherdevelopmental biology-based research is needed toidentify other tissue-specific biomarkers as well as‘‘normal’’ expression levels and kinetic changes of thesesoluble markers associated with adequate new boneformation and mineralization. The acquisition of thesetypes of data would enable the application of auto-mated feedback control loops into the bioreactor designas variations from normal expression levels may indi-cate the need for changes in frequency of mediumchanges, perfusion rates, or other cultivation parame-ters. Soluble molecules are not the only potential bio-markers. With further research, it may be possible touse imaging modalities to provide relevant biomarkersof tissue development.

4. Imaging compatibility. Noninvasive imaging can provideuseful information for correlating with biochemicalmarkers. It is therefore crucial that the bioreactor designbe compatible with specific imaging modalities so thatthe graft could be monitored during development. Animaging-compatible bioreactor would enable longitu-dinal evaluation of tissue growth without compromis-ing graft sterility.

Imaging compatible bioreactors have been designed by anumber of groups to accommodate a variety of imagingmodalities, including micro-CT,74,75 confocal microscopy,76

magnetic resonance imaging (MRI),77 nuclear magnetic res-onance (NMR),78 optical coherence tomography,79,80 andDoppler optical coherence tomography.81,82 In the case ofbone grafts three imaging modalities may prove most ben-eficial: micro-computed tomography (micro-CT), cone-beamCT (CBCT), and optical coherence tomography (OCT). Thereare relative advantages and disadvantages of each techniqueas well as potential limitations to its use in a clinically rele-vant bioreactor.

a. Micro-CT is a relatively cheap and high-resolutionimaging modality that has been used effectively toexamine small animals and bony scaffolds on a scaleof 1–50mm.83–87 The duration of a micro-CT scanvaries according to resolution, but can be as little as30 min.83 Micro-CT compatible bioreactors have al-

ready been developed by several groups.74,75 Ha-genmuller et al. designed a micro-CT-compatiblebioreactor for the purpose of monitoring the osteo-genic progress of cells in scaffold culture.75 In thissystem, scaffolds can be housed in bioreactor car-tridges made of polysulfone, a material with low ra-dio opacity, which allowed the scaffolds to bescanned via micro-CT without removal from thesterile cartridge housing. The cartridges could also beplaced into a mechanical stimulation unit (MSU) toprovide loading stimulation. Hagenmuller et al.found that this system allowed for the acquisition ofhigh-quality CT images of a bone biopsy sampleplaced in the cartridge that were comparable to im-ages obtained when the sample was housed in ascanning vial. There is no currently published data onthe use of this system to monitor mineral depositionover time; however, the cartridge approach and theability to add a mechanical stimulation componentmake this a potentially valuable system for thegrowth and monitoring of bone scaffolds. Anothergroup was able to examine the amount and rate ofmineralization of bone constructs cultured underperfusion by modifying the construct chamber ge-ometry to accommodate a standard micro-CT scan-ner.76 The reactor was sized such that it would fit intothe scanner and the wall thickness was reduced tomatch standard micro-CT chambers, demonstratingagain that designing an imaging-compatible biore-actor is technically feasible. Such baseline studiesmay establish ‘‘normal’’ parameters, such as miner-alization rate, that a graft should meet throughoutculture. The study also validated the efficacy ofthe approach by scanning twice versus once withina 5-week period and evaluating cell viability andmineral deposition. They demonstrated that scanningmultiple times did not negatively affect the graft inthe short term. However, given that the main concernwith micro-CT is the high radiation dose, it may benecessary to establish the long-term effects of micro-CT scanning on cultured constructs.

b. An alternative is cone-beam CT (CBCT), which al-lows for image acquisition using a single rotation,resulting in lower radiation (0.1–1 cGy vs. 10–100cGy for micro-CT)88–91 and faster scan times (on theorder of 1–3 minutes).92–94 This makes CBCT a po-tentially superior choice to micro-CT for scaffoldimaging as it is more amenable to multiple, longitu-dinal scans to provide insight into tissue develop-ment. The resolution of cone-beam CT, however, isconsiderably lower than that of micro-CT, making itsignificantly less quantitative.92 In their study com-paring CBCT to micro-CT, Loubele et al. reportedmicro-CT delivers, on average, a 20 times more effec-tive radiation dose than does CBCT,92 and has theadvantage of achieving much higher resolution (aslow as 1mm as opposed to 100–500mm for CBCT).92–94

A CBCT-compatible bioreactor has requirements andconsiderations similar to the one used for micro-CT76:modified chamber dimensions to allow for imagingwithout removing the scaffold and housing materialthat would not interfere with scans.

68 SALTER ET AL.

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c. Another possibility for imaging is optical coherencetomography (OCT), which is a light-based techniqueand therefore delivers no radiation. It can achieve1-mm resolution imaging up to a depth of around2 mm.95 OCT or Doppler OCT has already beenutilized in several perfusion bioreactor setups tomonitor tissue growth or flow.80–82 For example,Bagnaninchi et al. used an OCT-compatible perfusionbioreactor to assess cell seeding/growth and ECMdeposition in microchannels of a porous chitosanscaffold seeded with porcine tenocytes.80 In this case,the scaffold had a small enough diameter to allowOCT imaging through the clear sample chamber.Beyond the 2-mm penetration limit, as would be thecase with larger grafts, OCT imaging may utilize aneedle in order to provide increased penetration.96

OCT scanning using needles could quite conceivablybe interfaced using these perfusion needles withoutmuch need for altered bioreactor design. However, asis the case with soluble biomarkers, considerable re-search is required in order to correlate data derivedfrom this modality with quantitative measures ofbone growth and tissue maturity.

5. Ease of use. Much like MRI scanners and other high-techclinical tools, it is essential to design a bioreactor thatprovides sophisticated technology, while remainingintuitive and simple to operate. Operators in the clinic,such as nurses or technicians, would need minimaltraining to use the bioreactor. An easy-to-use devicealso reduces operator dependency, as no specializedskill is required for operation.

Estimated Cost of Bone Grafts

Even accounting for these scientific and practical consid-erations, the biggest challenge in translating bioreactor-basedapproaches to the clinical applications arises from financialconsiderations. The feasibility of bioreactor technology formedical use depends greatly on whether the cost of im-plementing such technology would outweigh the benefits.The prospective cost should also be compared to the costs ofcurrently available or developing cellular/scaffold technol-ogies. In addition, for bioreactors to become a useful clinicaltechnology we must identify and address the economicbottlenecks in bioreactor design and use. We have comparedthe major cost factors, scale-up considerations, and potentialeconomic bottlenecks of TE-based technologies for orthope-dic applications (Table 2). We also compare, when available,the cost of commercial products in these categories. In thecase of cell treatments, the table is also broken down ac-cording to how the cells are prepared for the procedure, i.e.,traditional flask culture, bioreactor culture, or no in vitrocultivation. Each of these options has their own economicconsiderations that will cause differences in costs.

Currently, the cost of using a tissue engineering (TE)scaffold can only be roughly estimated due to the progres-sive nature of large graft bioreactor technology. However,through careful evaluation of costs associated with bioreac-tor systems, cell supply, and current technologies (see Table2), we project a cost estimate for bioreactor-grown bonegrafts. The one-time cost for designing and building a bio-reactor system that meets GMP standards and incorporates

both biomarker monitoring and cell culture can be estimatedto be between $25,000 and 35,000.97–102 The eventual cost of agraft, however, takes into account the fact that a bioreactorwill be used multiple times and, as such, bioreactor costs areamortized over many grafts. For this analysis we have splitthe cost of the bioreactor over 10 grafts as a conservativeestimate, giving a base cost of $2500–3500 per bioreactor use.Cell costs range from $500 to 1500 depending on the numberof cells needed and the source (allogeneic or autologous). Inour analysis, we have set the cost for cells and media pergraft at $2000. Labor costs for a clinic-based system could bedrastically reduced by having an on-site bioreactor with asimple user-interface that can be run by hospital nurses ortechnicians and by automating much of the procedure. Forexample, media changes, biomarker sampling/testing, andmedia condition monitoring (pH, O2/CO2 content) can all beautomated. The cost of biomarker testing can be estimatedby looking at the current market costs of biomarker ELISAkits. If we look at the cost of testing for a bone formationbiomarker such as IL-11, a 96-well plate ELISA generallycosts $400–500103–105 for biochemical analysis. Another costfactor that can be included is the price of imaging the con-struct. One proposed imaging method is CBCT, which costsan average of $200–600 per scan,106,107 while the machineitself plus installation and personnel training costs about$150,000.108 Because CBCT can be used for many otherpurposes involving craniofacial diagnostics or surgeryplanning,109 and does not need to be a bioreactor-specificpurchase, we have only included the cost of individual scans(averaged at $400) in the bioreactor cost estimate. Assumingthe graft can be imaged and assayed for biochemical markersat weekly intervals, we can incorporate these factors bymultiplying the cost per assessment by weeks cultured (inthis analysis we assume a highly conservative estimate of 4–8weeks of culture). If we take into account the per-graft costs(assays, imaging, cells, and media), we come to an estimateof about $4000–$6000. The rest of the graft cost will comefrom the bioreactor and labor. An additional $5000 is in-cluded to cover labor costs and other factors that have notexplicitly been included in the estimate, such as the cost ofoccasional graft failure to grow or contamination. Taking allof this into account, we project that the most conservativeestimate for a bioengineered graft would likely fall into therange of $10,000–15,000. We can compare this range againstcurrent technologies to see if it is an appropriate estimate.With many of the other factors being the same, we reasonthat the estimated cost of the engineered bone graft shouldfall below that of similar cell-based procedures that utilizecentralized, off-site facilities. However, a bioengineered graftwould be more expensive than simply using a donor bonegraft or a suspension of bone marrow/mesenchymal stem cells(without culture). Using current technology prices as lowerand upper bounds for a reasonable range (Table 2),110–123 itappears that we have arrived at a reasonable ballpark rangefor the estimated cost of a bioreactor grown bone graft.

Although the projected cost of a tissue engineered graftaligns well with that of other cell-based approaches, it issignificantly more expensive than a standard bone graft.Hence, it is important to consider under what conditions apatient would consider the increased cost justifiable. This isknown as the headroom analysis and is another crucial com-ponent of feasibility assessment.124 Tissue-engineered bone

BONE TISSUE ENGINEERING BIOREACTORS 69

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70

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grafts may be reasonably considered when donor site mor-bidity associated with harvesting an autograft is a significantconcern and allograft resorption and low integration typi-cally present a problem. One such example of a niche marketis spinal fusion surgeries, especially in the lumbar region. Inthese cases iliac crest harvesting for autografts often leads tolong-lasting pain in a significant number of patients under-going this surgery.125,126 A recent study found that a groupof patients who underwent posterior lumbar spinal fusionusing an iliac crest autograft as a bone graft source experi-enced a significant amount of pain and functional limitationeven at 12 months after the surgery.120 In addition, allograftsare disadvantageous in the case of posterior lumbar fusionsand often lead to incomplete integration with host bone andresorption.126–129 Tissue engineered grafts could also beuseful in cases of large defects when there is not enoughautogenic material to fill the defect and allograft material hasshown previous problems of resorption due to limited vas-cular infiltration. While surgical vascularization of largergrafts is helpful in this regard, it is also a technically de-manding, expensive procedure that could be avoided byusing prevascularized tissue engineered grafts.130,131 Tissueengineered bone grafts may be particularly relevant in cra-niofacial applications where the gross geometry is crucial tofunction and the current best option requires calvarial boneharvesting.132 Bone grafts could be engineered in the precisesize and shape needed for cranioplasty, thus eliminating theneed for bone harvest and eliminating aesthetic concerns.Thus, although the market for bone engineered grafts will besmaller due to their high cost in comparison to normal allo/autografts, it is our assessment that such a technology couldprosper in cases in which normal grafts are unsuitable orhave a high chance of failure.

Summary: The Application of Tissue-EngineeredBone Grafts

Currently, several multidisciplinary efforts are being madeto improve the size and quality of the bone grafts that can begrown in vitro. These efforts focus largely on aspects of celltype, scaffold structure and composition, or cultivation pa-rameters in various bioreactor systems. However, unlessthere is holistic consideration for how these engineeredproducts can be employed in clinical settings, these studiesrun the risk of being practically and commercially nonviablefrom the onset. The past two decades have provided awealth of lessons regarding the eventual feasibility of cell-based therapies. Chief among these lessons has been thatcommercial viability can trump science in determiningwhether an approach will become widely applicable.133

Consequently, in determining whether bone bioreactors willfacilitate the clinical application of engineered bone grafts,we must consider clinical scenarios in which engineeredgrafts would be desirable, the scientific feasibility of usingengineered grafts relative to cell-seeded scaffolds, and eco-nomic aspects. The clinical need for engineered grafts, par-ticularly in cases in which autografts are unavailable, hasbeen firmly established and our analysis indicates that thecost of this approach lies within the range of other cell-basedtherapies. Therefore, many more in vivo experiments in smalland large animal models need to be performed to robustlyestablish and quantify the benefits of using engineered grafts

relative to other approaches so that these can be weighedagainst the costs of additional bioreactor cultivation.

Acknowledgments

This work is supported by a grant from the Department ofDefense (DM090323). The authors would also like to ac-knowledge Dr. Lynne Jones for her help in cost research andinsight into current use of bone grafts technologies in or-thopedic medicine.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Warren L. Grayson

Laboratory for Craniofacial and Orthopaedic Tissue EngineeringDepartment of Biomedical Engineering

Johns Hopkins University400 N. Broadway

Smith 5023Baltimore, Maryland 21231

E-mail: [email protected]

Received: April 11, 2011Accepted: September 8, 2011

Online Publication Date: December 21, 2011

BONE TISSUE ENGINEERING BIOREACTORS 75

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