2000 karen burg

*Corresponding author.

Biomaterials 21 (2000) 2347}2359

Biomaterial developments for bone tissue engineering

Karen J.L. Burg!,*, Scott Porter", James F. Kellam"

!Department of Bioengineering, Clemson University, 501 Rhodes Engineering Research Building, Clemson, SC 29634-0905, USA"Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC 28232-2861, USA

Abstract

The development of bone tissue engineering is directly related to changes in materials technology. While the inclusion of materialsrequirements is standard in the design process of engineered bone substitutes, it is also critical to incorporate clinical requirements inorder to engineer a clinically relevant device. This review presents the clinical need for bone tissue-engineered alternatives to thepresent materials used in bone grafting techniques, a status report on clinically available bone tissue-engineering devices, and recentadvances in biomaterials research. The discussion of ongoing research includes the current state of osseoactive factors and the deliveryof these factors using bioceramics and absorbable biopolymers. Suggestions are also presented as to the desirable design features thatwould make an engineered device clinically e!ective. ( 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Absorbable; Bone morphogenetic protein; Bone tissue engineering; Ceramic; Demineralized bone matrix; Polymer

1. Introduction

1.1. Rationale for bone tissue-engineering

There are multiple clinical reasons to develop bonetissue-engineering alternatives, including the need forbetter "ller materials that can be used in the reconstruc-tion of large orthopaedic defects and the need for ortho-paedic implants that are mechanically more suitable totheir biological environment. The traditional biologicalmethods of bone-defect management include autograft-ing and allografting cancellous bone, applying vas-cularized grafts of the "bula and iliac crest, and usingother bone transport techniques. Although these are thestandard treatments, shortcomings are encountered withtheir usage. Since bone grafts are avascular and depen-dent on di!usion, the size of the defect and the viability ofthe host bed can limit their application. Furthermore, thenew bone volume maintenance can be problematic dueto unpredictable bone resorption. In large defects, thegrafts can be resorbed by the body before osteogenesis iscomplete [1,2]. Not only is the operating time requiredfor harvesting autografts expensive, but often the donortissue is scarce, and there can be signi"cant donor sitemorbidity associated with infection, pain, and hematoma

[3}7]. Allografting introduces the risk of disease and/orinfection; it may cause a lessening or complete loss of thebone inductive factors [8]. Vascularized grafts requirea major microsurgical operative procedure requiringa sophisticated infrastructure. Distraction osteogenesistechniques are often laborious and lengthy processes thatare reserved for the most motivated patients [9,10]. An-other method of bone defect repair is via bone cement"llers. Bone cements are prepared in the operating roomand therefore can be susceptible to infection.

Bone marrow replacement is another possible tissue-engineering application for the treatment of patientsfollowing high-dose chemotherapy and/or radiationtreatment [11]. The acquisition of bone marrow requiresthe sterile aspiration of the marrow from the posterioriliac crest. Marrow may be used in tissue-engineeringculture and speci"cally as a basis for bone marrow ex-pansion. The progenitor cells can be cultivated and se-lected as needed.

Bone tissue engineering may potentially provide alter-native solutions that possess better mechanical proper-ties than those used currently. This may decrease thevascular insult of the implant to the bone and causeless-stress shielding, perhaps decreasing the incidence ofimplant-related osteopenia and subsequent refracture.The mechanical properties of a bone tissue-engineeredconstruct could be modulated to avoid such sequelae.A related application is the use of a tissue-engineered

0142-9612/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 1 0 2 - 2

implant surface to permanently stabilize implants bycoating the prosthesis with cells or tissue before im-plantation. This could be extremely useful in reconstruc-tive orthopaedic surgeries that potentially have highincidences of failure secondary to large bone defects [12].

Bone regeneration requires four components: a mor-phogenetic signal, responsive host cells that will respondto the signal, a suitable carrier of this signal that candeliver it to speci"c sites then serve as a sca!olding forthe growth of the responsive host cells, and a viable, wellvascularized host bed [13,14]. Bone tissue engineering,for the purpose of this review, is the use of a sca!oldingmaterial to either induce formation of bone from thesurrounding tissue or to act as a carrier or template forimplanted bone cells or other agents. Materials used asbone tissue-engineered sca!olds may be injectable orrigid, the latter requiring an operative implantation pro-cedure. To this end, the areas of materials research can begenerally divided into acellular and cellular, with drugdelivery overlapping in both areas.

The "rst part of the research discussion in this reviewwill focus on acellular systems. Acellular will be classi"edas materials on or in which no additional cellular com-ponent is cultured. The discussion will include materialsthat are clinically available as well as those materials thathave potential clinical use and are in the early stages ofresearch. Acellular materials will be regarded as solid,absorbable "llers that will disappear over time, or poroussca!olding that immediately allows room for bonegrowth into the construct. The second part of the re-search discussion will focus on cellular systems. Thecellular materials are classi"ed, for this review, as scaf-folds to which a cellular component is added prior toimplantation.

The third part of the research discussion will focus onmaterials designed for drug delivery. The use of growthfactors has a potential to markedly increase sca!olde!ectiveness. Transforming growth factor b and themore recently discovered bone morphogenetic proteins(BMPs) are integral in the regulation of embryologicbone formation and post-traumatic bone healing/forma-tion [13,15}17]. Researchers have demonstrated thatBMP can cause bone formation de novo if present insu$cient quantity [18]. BMP has been delivered to local-ized sites in order to repair bone defects and nonunionsin several di!erent experimental models [19}33].

The materials commonly used in all three approachesare ceramics, polymers or composites. The ceramics andpolymers are either absorbable or nonabsorbable, andthe polymers can be naturally derived materials orsynthesized materials. Bone tissue-engineering systemshave included demineralized bone matrix, collagencomposites, "brin, calcium phosphate, polylactide,poly(lactide-co-glycolide), polylactide-polyethylene glycol,hydroxyapatite, dental plaster, and titanium [13,34}38].

2. Approaches toward bone tissue engineering

2.1. Desired features of a bone tissue-engineering material

Before considering the desired features of potentialtissue-engineering materials, it is useful to understandtwo concepts of bone regeneration for tissue-engineeringconstructs, speci"cally osteoconduction and osteoinduc-tion. Osteoinduction is de"ned as the ability to causepluripotential cells, from a nonosseous environment todi!erentiate into chondroctyes and osteoblasts, culmina-ting in bone formation [39}41]. An osteoinductive ma-terial allows repair in a location that would normally notheal if left untreated [8]. Osteoconduction supports in-growth of capillaries and cells from the host into a three-dimensional structure to form bone. An osteoconductivematerial guides repair in a location where normal healingwill occur if left untreated.

Several years ago, researchers became aware that theosteoconductive properties of the synthetic absorbablepolymers were dependent on their location and the struc-ture of the polymer [42]. The tubular absorbable poly-mers used in long bone defects, for example, are believedto promote bone growth by excluding the surroundingsoft tissue and its undesired cellular elements from thedefect, by maintaining an osteogenic-rich medullary envi-ronment within the defect, and by allowing direct bonegrowth onto the polymer skeleton [42}45]. Polymers candi!er in their molecular weight, polydispersity, crystal-linity, and thermal transitions, allowing di!erent absorp-tion rates. Their relative hydrophobicity and percentcrystallinity can a!ect cellular phenotype. Variations insurface charge will a!ect cellular spreading or a$nity forthe surface, which can also cause changes in phenotypicexpression [46].

Local tissue responses to polymers in vivo depend onthe biocompatibility of the polymer as well as its degra-dative by-products [47]. The mechanism of erosion canalso a!ect the pH of the surrounding environment andsubsequent response. The absorbable polyesters, forexample, are largely hydrolysed through bulk erosion[48}50]. Although early absorption times may demon-strate a stable pH [51], there is the potential for a suddendecrease in local pH after a prolonged absorption time inslower absorbing systems. Oxygen tension will also a!ectthe type of cell that proliferates; therefore, the correctenvironment is very important [52].

Poly(lactide-co-glycolide) matrices are often used asconstruct materials. They can be custom synthesized tomeet an absorption time requirement, and they are alsoclinically familiar. There are several methods of process-ing these porous, synthetic matrices. The most commonmethod is solution cast, particulate-leached as developedby Mikos [53]. In order to re"ne this method, it is quitepossible to modulate the pore topography and size tosuit a particular cell type, e.g., osteoblasts. Work in our

2348 K.J.L. Burg et al. / Biomaterials 21 (2000) 2347}2359

Fig. 1. (a,b) Bone topography (40]magni"cation) varies according to function and location. Photographs courtesy of L. Jenkins, HT (ClemsonUniversity Department of Bioengineering). (c,d) Polylactide sca!old topography (20]magni"cation) may be varied by processing technique.

laboratory [54] as well as by others [55] has shown thatthe pore shape can have a profound e!ect on the attach-ment and long-term survival of cells on a surface. Ourwork has shown that, for a speci"c cell type, there is anoptimal pore topography that can be readily modulatedby careful selection of porogen. Since bone has verydi!erent structures depending on its function and loca-tion, it stands to reason that the same pore shape maynot be ideal for all potential uses. Fig. 1 reinforces thispoint by demonstrating di!erent appearances of boneand di!erent polymer sca!old topographies.

Pore size and tortuosity can be carefully modulated tocontrol the release of a material complexed to the poly-mer [56,57]. Pore size is also very cell-type speci"c.Gogolewski's laboratory reported that, although poly-ester membranes with pore sizes up to 200 lm diameterpromoted bone growth within a 1-cm defect of the radiiof rabbits, smaller pore sizes promoted the most growth[43]. Tsurga and coworkers have suggested that theoptimal pore size of ceramics that supports ectopic boneformation is 300}400 lm [38]. Holmes similarly sugges-ted that the optimal pore range is 200}400 lm with theaverage human osteon size of approximately 223 lm[58].

Porosity can, however, adversely a!ect importantmechanical characteristics of a polymer, requiring morecomplex material designs. Increased hydroxyapatite(HA) porosity, for example, decreases its malleability andreduces its ability to conform to the irregular surfacesthat may be present in host bone [26,59,60]. Continuityof the host bone/polymer interface is essential for oss-eointegration [26]. Koempel and coworkers determinedthat re-engineering a porous HA to deliver rhBMP 2 re-sulted in nearly uniform "xation. The authors postulatedthat the porous HA acted as a conduit for the BMP-induced growth of new bone allowing better "xation ofthe HA composite despite its porosity [28].

Osteoblast proliferation is sensitive to surface topogra-phy [52], strain or other mechanical stimuli. The aspectratio of a material will in#uence the bony or "broblasticdi!erentiation of a tissue. Particle size, shape, and surfaceroughness a!ect cellular adhesion, proliferation, andphenotype. Cells can discriminate even the subtlest cha-nges in topography, and they are most obviously sensi-tive to chemistry, topography, and surface energy. Suchsurface features are particularly interesting when consid-ering an absorbable material, since this is a dynamicmaterial, always presenting a new surface. Additionally,

K.J.L. Burg et al. / Biomaterials 21 (2000) 2347}2359 2349

Table 1Select critical consideration in bone tissue-engineering sca!old design[27,58,62]

Desirable qualities of a bone tissue-engineering sca!old

Available to surgeon on shortnotice

Promotes bone ingrowth

Absorbs in predictable mannerin concert with bone growth

Does not induce soft tissue growthat bone/implant interface

Adaptable to irregular woundsite, malleable

Average pore sizes approximately200}400 lm

Maximal bone growth throughosteoinduction and/orosteoconduction

No detrimental e!ects to surround-ing tissue due to processing

Correct mechanical andphysical properties forapplication

Sterilizable without loss ofproperties

Good bony apposition Absorbable with biocompatiblecomponents

the surface energy may play a role in attracting particularproteins to the surface of the material and, in turn, thiswill a!ect the a$nity of the cells to the material. Table 1is a summary of several critical considerations in thedesign of a bone tissue-engineering material. Brekke [61]provides a detailed list of desirable device characteristicsof a manufactured bone graft substitute.

3. Bone tissue-engineering materials research

3.1. Acellular systems

3.1.1. Acellular systems: naturally derived polymersIn the early 1960s, Urist and several co-workers realiz-

ed that demineralized bone consistently induced boneformation in ectopic tissues of experimental animals[63}65]. A hydrochloric acid extraction process decalci-"ed the bone matrix [64,66,67], producing demineralizedbone matrix (DBM), a compound that was later shown topossess inherent osteoconductive and osteoinductiveproperties [66]. Recent research has proven that corticalbone is the preferred choice for DBM synthesis as it ismore osteoinductive with a lower antigenic potentialthan cancellous bone [68]. Urist described the histologi-cal process by which new bone is formed under thein#uence of DBM in ectopic sites [66]. The tissue isinitially in"ltrated by in#ammatory and mesenchymalcells. Early angiogenesis, progenitor cells, and chon-drocytes can be appreciated by 3 weeks. Shortly there-after, osteoblasts, osteocytes, and chondrocytes appear tolead to the synthesis of cartilage which is transformedinto woven bone over the next several weeks. By 4 weekspost-implantation, osteoclasts and bone remodeling cellsare present, and the bone marrow is formed at about 4}6weeks [14,66]. This process recapitulates the process ofendochondral ossi"cation.

Russell and Block point out that the processing of theDBM can greatly in#uence the "nal osteoinductive abil-ity [69]. Ethylene oxide, the sterilization agent for manysynthetic, absorbable devices [70], can render the DBMcompletely devoid of osteoinductive potential, althoughthe osteoconductive potential would remain [69]. Inaddition, they assert that ethanol is e!ective in reducingthe bacterial load without a!ecting the osteoinductivepotential of the matrix.

There has been a persistent orthopaedic interest inDBM because of its therapeutic potential in the treat-ment of bony defects, nonunion, and its application injoint fusion procedures. Russell and Block evaluated 21studies that used DBM in the treatment of a variety ofclinical and experimental orthopaedic situations thatwould have normally warranted treatment with standardbone graft [69]. Even though the sterilization proceduresvaried, they determined that more than 80% of theauthors reported favorable results with the use of DBMregardless of the level of di$culty of the cases [69].Tiedeman and coworkers were able to clinically demon-strate a 77% (30 of 39 patients) union rate for a series ofpatients with non-unions, arthrodeses, acute fractureswith bone loss or comminution, and osseous or cavitarydefects that were treated with DBM plus autogenousbone marrow aspirate [3]. Excluding the patients whohad a documented non-union before the study's incep-tion, DBM increased the union rate for the remainingpatients to 90% (26 of 28 patients). These rates wereobtained despite the sterilization of DBM with ethyleneoxide [3].

Gepstein and coworkers examined the ability of DBMto heal large long bone defects in rats [4]. In their study,defects that were greater than 50% of the length of theradial diaphysis were created in the bilateral front limbsof 33 rats. At 21 days postoperatively, densitometrymeasurements of the defect treated with DBM were with-in 91% of the native ulna for the 27 surviving rats. By 35days, 71% of the rats had radiographic union, and theother 29% had at least union of one end of the defect [4].Urist and Daws obtained a low pseudarthrosis rate of12% in their series of 40 patients with intertransverseprocess fusion using autologous bone graft from thespinous processes. The graft was harvested during thesurgical approach and supplemented with DBM. Thisrate compared favorably to the reported rate in the bestseries using iliac crest bone graft at that time. Further-more, Urist and Daws were able to perform multilevelfusions without the increase in morbidity common inthese procedures when the bone graft source is the iliaccrest [68].

MaK rtson's investigations of the use of viscose cellulosesponge determined that it too is useful for bone tissueengineering. Sponges placed in the femoral shafts of ratsshowed that primarily osteoconduction occurred, sincebone formation was predominantly at the periphery of


the sponges. No microstructural or physicochemicalcharacteristics of the sponges were given.

3.1.2. Acellular systems: synthetic polymersSynthetic injectable materials are "nding appeal as

bone tissue-engineered sca!olds, due in part to theirminimally invasive implantation. Elissee! and coworkersdeveloped photopolymerizable materials that can be in-jected as liquids and photopolymerized to localize thematerial [71]. Although they have tested this for trans-dermal application, with the increased interest in minim-ally invasive procedures, this may have orthopaedicapplication as well.

Synthetic matrices have also been assessed as acellularbone tissue-engineering materials. Gogolewski's groupused a poly-L-lactide (PLLA) membrane to cover 1-cmdefects that they created in the radii of skeletally maturerabbits [45]. They chose a polymer with pore sizes of5}15 lm diameter, a thickness of 250 lm, and an in vivolife span of 18}24 months. At the conclusion of the study,they had grossly and histologically demonstrated thatcortical bone had regenerated to span the defect [45]. Inanother study by Gogolewski's group, they used theYucatan pig model to create a critical defect of 25% ofthe length of the radius [72]. The defects were coveredwith a PLLA or a poly-L-co-D,L-lactide (PLDL) mem-brane or with a PLLA or PLDL membrane that hadbeen synthesized with calcium carbonate in order todecrease the relative amount of polymer in the mem-brane. They demonstrated that the membrane facilitatedthe rapid formation of new bone growth without adversereactions. Importantly, the addition of calcium carbonatedid not change the dynamics of the membranes eventhough the amount of actual polymer within them wasreduced by 50% [72]. Gugala and Gogolewski failed toinduce signi"cant bone formation when they used mem-branes with a pore size of 10}20 lm to cover 4-cm defectsin the tibiae of sheep [44]. They concluded that bonedefects that are greater than a critical size will not heal,even if the synthetic membranes are used to facilitatebone regeneration. When they used cancellous bone graftin conjunction with synthetic membranes in the samesized defect, they were able to induce signi"cant bonehealing. They postulated that, in addition to theaforementioned roles synthetic membranes might play,they could also optimize the contact between the softtissues and the bone graft and avoid the graft's excessiveresorption [44].

Another possible "ller material is poly-e-caprolac-tone-co-lactide. This has been observed in nonosseousapplications [73,74] but is newly applied to bone tissueengineering as a paste or wax. Ekholm and coworkersevaluated the absorption and biocompatibility of thiscopolymer using a rat femoral defect model [75]. Thecopolymer was speci"cally a paste of 40 : 60 poly-e-cap-rolactone-co-D,L-co-L-lactide with a 50 : 50 D-lactide to

L-lactide ratio. The material elicited a moderate in#am-matory response in bone and was still present after 1 yr inthis preliminary study.

Photocrosslinkable polyanhydrides are new materialsthat present certain advantages in orthopaedic applica-tions [76]. They absorb via surface erosion and thereforeare not susceptible to sudden losses in mass or loaddumping in delivery applications. The photopolymeriz-able element adds the potential for microfabrication ofporous sca!olds but also could allow an injectable ma-terial that can be subsequently crosslinked. Initial mech-anical studies show that these polymers demonstrateenhanced mechanical integrity [76].

3.1.3. Acellular systems: compositesPolymer composites with ceramic "llers have also been

investigated. Peter and coworkers reported a method inwhich poly(lactide-co-glycolide) constructs formed bysolvent casting/particulate leaching were crushed andthen compression molded with hydroxyapatite (HA) inorder to improve compressive yield strength [77]. Mikosreports a poly(propylene fumarate) (PPF) biodegradablebone cement that can be combined with a leachablecomponent and injected into osseous defects [78,79]. Thepolymerization of the material itself can be adjusted tocause foaming through the release of carbon dioxide,thereby producing a porous sca!old [80]. The injectablenature of the material allows it to "ll irregular osseousdefects, and the leachable component allows room forbone ingrowth (Fig. 2). This particular material also haspotential as a drug delivery system. Composite systemscan be formed with tricalcium phosphate (TCP) to im-prove mechanical integrity [81]. Similarly, Bennettshowed that a poly-dioxanone-co-glycolide based com-posite reinforced with HA or TCP can be used as aninjectable or moldable putty [82]. During the crosslink-ing reaction following injection, carbon dioxide is re-leased allowing the formation of interconnected pores.The carbon dioxide also causes expansion of the materialand essentially a press-"t, seamless interface.

Zhang and Ma prepared PLLA/apatite composites bysoaking porous PLLA constructs in simulated body #uidto allow development of apatite throughout the sca!old[83]. Apatite formation was enhanced by hydrolysis andnucleation, and both size and frequency was correlated toamount of exposed surface area. This may be of directinterest as a bone tissue-engineering sca!old or indirectlyin assessing currently existing sca!olds. Zhang and Mahave also examined PLLA/HA constructs formedthrough a standard polymer processing technique, ther-mally induced phase separation (TIPS) [84]. As withother absorbable systems formed in this manner [85],the polymer microstructure can be readily controlledthrough manipulation of the component concentrationsas well as the processing temperature and cooling rate.


Fig. 2. (a) Scanning electron micrograph of PPF sca!old: 10 wt% PPF,90 wt% porogen; porogen removed with water leaching after PPFcrosslinking. (b) Scanning electron micrograph of PPF sca!old: 30 wt%PPF, 70 wt% porogen; porogen removed with water leaching afterPPF crosslinking. Micrographs courtesy of Dr. A.G. Mikos (RiceUniversity Department of Bioengineering).

3.1.4. Acellular systems: ceramicsThe absorbable, inorganic materials that have been

investigated include CaCO3

(argonite), CaSO4~

2H2O

(plaster of Paris), and Ca3(PO

4)2

(beta-whitlockite,a form of TCP) [47]. The most widely studied calciumphosphate ceramics are TCP, HA (Ca

10(PO

4)6(OH)

2),

and the newest tetracalcium phosphate [47,59]. The ap-peal of the calcium phosphates rests largely with theirbiocompatibility. Since they are protein free, minimalimmunologic reactions, foreign body reactions, or sys-temic toxicity have been reported with their use [47,86].Although the inorganic ceramics have not shown os-teoinductive ability, they certainly possess osteoconduc-tive abilities as well as a remarkable ability to binddirectly to bone [47,86].

Friedman and coworkers have created a new tetracal-cium phosphate that addresses the di$culties with mal-leability that can be encountered with the high-porosityceramics [59]. BoneSourceTM is a hydroxyapatite that issupplied in a powder. When it is mixed with sterile water,it is changed into a conformable, paste-like consistency.When it sets, it does so with a microporous structure of8}12 lm. Despite its microporous structure, the authorsnote that, unlike older ceramic HA implants, Bone-SourceTM is rapidly adherent to bone and possesses theunique quality of direct conversion to new bone withoutloss of implant volume. They have termed this processosteoconversion [59]. In a study of 103 patients withcranial defects in which BoneSourceTM was used, thesuccess rate, which was based on maintenance of theimplant and implant volume at 24 months, was approx-imately 97% [59]. Interpore 200 is a coralline-derivedporous HA trabecular-like structure with average poresizes of 200 lm. Hydroxyapatite is a very slowly de-graded material and therefore can be fashioned into anappropriate shape and prefabricated as a vascularizedbone #ap. Levine successfully showed the potential ofthis system in rabbits [27].

Ceramic processing is also advancing with the devel-opments of photopolymerizable biopolymers. Garg andcoworkers leveraged stereolithography technology tofabricate ceramic constructs using a concentrated col-loidal dispersion in an aqueous photocurable polymersolution [87]. This has potential for controlling pore sizeand porosity and therefore precision fabrication of por-ous templates.

3.2. Role of bone morphogenetic protein (BMP) systemsin bone tissue engineering

Near the time that Urist and coworkers "rst generateddemineralized bone matrix, they proposed that its os-teoinductive properties were caused by a novel factor[39]. Several years later, Urist and Strates named thisfactor bone morphogenetic protein (BMP) and, in con-junction with several other coworkers, puri"ed rat andrabbit BMP [67,88,89]. Since then, several groups ofinvestigators have shown that BMP is actually a group ofproteins responsible for a variety of events in embryogen-esis and in the postnatal skeleton [13,17].

BMPs are of particular interest to the "eld of ortho-paedics because of the critical role that they have beenshown to play in embryological bone formation, osteo-induction, and bone repair as well as the possibility thatthey may assist in the replacement of the standard auto-genous bone graft [15,17,25,33,35,90,91]. To date, 15BMPs have been characterized and cloned [13]. Wangand Sampath demonstrated that recombinant humanBMP (rhBMP) 2 and 7, respectively, are capable ofinducing bone formation in a process that mimicsendochondral ossi"cation at ectopic sites in a rat model


[90,92]. Using rats, Einhorn demonstrated that the nor-mal pathway of healing for fractures could be acceleratedwith the percutaneous injection of rhBMP 2 [35,93].Bostrom and coworkers were able to demonstrateintense staining of fracture calluses with the use ofanti-BMP 2 and four antibodies. They were able todemonstrate an almost biphasic intensity of staining thatrelated to a primary intensity within the primitive mesen-chymal and chondrocytic cells, and a second period ofintensity within osteoblasts as they invaded the cartilagi-nous callus [15].

Though the use of BMPs may prove to be importantto orthopaedic surgery as a whole, it is evident thatBMPs could be invaluable in orthopaedic reconstructivesurgery. BMP has been delivered to localized sites inorder to repair bone defects and nonunions in severaldi!erent experimental models [19}33]. Delivery systemshave included demineralized bone matrix, collagencomposites, "brin, calcium phosphate, polylactide,poly(lactide-co-glycolide), polylactide-polyethylene glycol,hydroxyapatite, dental plaster, and titanium [13,34}38,94]. An ideal delivery system would allow a slow releaseof the BMPs, be biologically and immunologically inert,quickly absorbed, and supportive of cell proliferationand angiogenesis. It would also possess enoughrigidity to withstand deforming forces until absorbed.Lastly, it would be easily stored, handled, and sterilized[13,14].

3.2.1. BMP systems: naturally derived polymersIn long bones such as the tibia, the existing treatment

options for large segmental defects are usually limited tomultistaged reconstruction and/or amputation [95]. Sev-eral authors have demonstrated that BMP 2 and 7,delivered to clinical and experimental osseous defects upto 17 cm in length, have signi"cantly and favorablya!ected the ability of these defects to heal [13,18,24,25,33,35,96}99]. Yasko and coworkers created 5-mm de-fects in the femora of 45 adult rats [33]. By showinga 100% union rate using a combination of rhBMP 2and DBM as a carrier, they concluded that BMP mightprove to be a bone graft substitute of unlimited quantity[33].

Reddi and Levine both cite insoluble collagen as apotential carrier for BMP, but point out that data arelimited for this matrix [27,56]. This may be, in part, dueto low compression strengths of constructs of this type.In an isolated study comparing delivery from collagenmatrices versus HA, TCP, glass beads, and polymethyl-methacrylate, the collagen was superior as a drug-releas-ing matrix [56].

In a series of six patients who had undergone severallower extremity procedures to gain soft tissue controlfollowing an acute traumatic event, Johnson andcoworkers achieved union in "ve of six patients withtibial defects that spanned 3}17 cm [25]. The authors

used autologous bone graft supplemented with hBMPand induced union with a single operative procedure. Inanother series, these same authors gathered 25 patientswith resistant nonunions that included partial or com-plete segmental bone defects [99]. Twenty-three of thesepatients had an average of three prior surgical proced-ures that failed to promote union of the bony ends.Within 3}7 months of treatment with hBMP and a DBMderivative as an onlay or inlay graft, 20 of the 25 patientsachieved union. Four of the remaining "ve patientsobtained union after a second procedure [99].

Although BMPs may represent an integral componentin the future of orthopaedic reconstructive surgery, prob-lems potentially exist with the types of carriers currentlyin use. The crude extract of BMP is hydrophobic, but asthe protein is puri"ed, it becomes more hydrophilic. Theimplantation of puri"ed, hydrophilic BMP promotestheir dispersion shortly after implantation and beforeosteoinduction can occur [100}102]. To prevent the dis-persion and to deliver the BMPs to the desired sites, theyare normally complexed to carriers that allow a slowrelease. Most of the protein-derived carriers such asDBM and the collagen composites have the potential ofantigenicity and exposure of the recipient to viruses andother infectious agents [3,13,17,25,68,96,97,99].

3.2.2. BMP systems: synthetic polymersAlthough select synthetic polymers such as PLLA can

also pose design issues due to in#ammatory responses orextended absorption times [21,43,44,51,94,100], investi-gators have examined the possibility of using syntheticpolymers as vehicles to allow the targeted delivery ofBMPs [21,47,94,100,103]. Ferguson and coworkersshowed that experimentally created trephine defectscould be successfully treated with puri"ed bovine BMP[21]. They complexed the bBMP with a poly-lactide-co-glycolide (PLGA) in one animal. This yielded unabsor-bed polymer surrounded by new bone as late as 16 weeksinto the experiment. The authors concluded that long-lasting copolymers might provide a barrier to completebone formation. Moreover, they postulated that morequickly degrading polymers could be used to aid in thecontrolled delivery of BMP to defects without interferingwith bone formation [21]. Kirker-Head and coworkersused rhBMP 2 combined with poly-D,L-lactide-co-glycol-ide (PDLGA) particles of 150}500 lm and autogenousblood to heal 2.5-cm defects in the femora of "ve of 10sheep. In the sheep that demonstrated union, the polymerwas completely absorbed and woven and lamellar bonebridged the defect [103]. Miyamoto evaluated severalpolylactic acids (PLAs) and polylactides of varying mo-lecular weights for their potential use as a carrier [100].These polymers were synthesized using polycondensa-tion and ring-opening techniques, respectively. The dif-fering polymerizations, and therefore molecular weights,allowed a variety of appearances of the "nal PLA/BMP


or PLLA/BMP complex that ranged from viscous liquidto pellets. It also allowed for di!ering lengths of timenecessary for complete degradation. The BMP used inthe experiment was puri"ed from rat osteosarcomas.Among PLAs or PLLAs of di!ering molecular weights,they determined that only PLA with a molecular weightof 650 daltons allowed BMP to reproducibly induce boneformation at ectopic sites (i.e., rat muscle belly). Further-more, by 2 weeks, the polymer was completely degraded[100]. In a follow-up study, the authors demonstratedthat when they complexed the 650-dalton PLA/BMPcomposite with a 200-dalton polyethylene glycol (PEG),they induced bone formation and the formation ofhaematopoietic marrow 3 weeks after implantation ofthe complex into rat muscle bellies [94]. The authorsalso noted twice as much bone formation in the ratsthat had been given the PLA/PEG/BMP composite.With the addition of hydroxyapatite, this compositewas converted from a viscous liquid to a doughypaste. The authors postulate that the viscous compositecould be used as an injectable osteoinductive materialwhile the doughy composite could be used as a matrix[94].

Not only does the molecular weight a!ect the BMPdelivery, so does the shape of the biomaterial deliveringBMP [56]. Reddi compared BMP loaded beads anddiscs of HA and observed that the beads were inactive,highlighting the need to customize the structure to suitthe speci"c application.

3.2.3. BMP systems: ceramicsKoempel and coworkers suggest that the addition of

BMP to a porous HA, of 200}500 lm pore size, wouldaugment the ingrowth of host bone [26]. Tsuruga andcoworkers [38] made similar observations by systemati-cally comparing a series of "ve BMP-releasing HAs, eachwith a di!erent pore range. They found that 300}400 lmyielded the highest bone formation. Takahashi andcoworkers demonstrated 100% fusion in 14 goats thatunderwent anterior multilevel cervical spine arthrodesiswhen they used synthetic porous HA and rhBMP 2 [32].Despite the lack of postoperative immobilization and theshortcomings of porous HA (the brittleness, the slowdegradation, the lack of osteoinduction, and the de-creased mechanical strength), the authors still demon-strated a 100% solid fusion rate with high-dose BMPand HA [32]. Asahina and coworkers demonstrated su-perior bone induction in comparison to controls when anHA/collagen/bovine BMP composite was placed intosurgically created mandibular defects in a primate model[19]. Gao and coworkers showed that a sheepBMP/collagen composite demonstrated osteoconductiveand osteoinductive properties when applied to an HAtubular construct with a pore size of 200}400 lm [22]. Intheir model of tibial defects in sheep, they reconstructedthe defects with the BMP composite, and the new bone

was characterized by more intense callus formation andincreased mechanical strength as compared with theircontrol groups [22].

4. Cellular systems

4.1. Cellular systems: naturally derived polymers

The collagen materials have also been applied as cellu-lar sca!olding systems. As collagen possesses no inherentstructural mechanical properties, engineering modi"ca-tions may be useful to provide a sti!er polymer to assistin force transmission of bone during the regenerativephase of healing. Yaylaoglu and coworkers demon-strated that porous collagen foams could be treated withcalcium solution to allow the deposition of calcium phos-phate and improvement of mechanical integrity [104].This technique shows promise in chondrocyte cultureand has great potential for bone application as well. It isnot known whether the collagen-based system has long-term stability in culture. Du and coworkers have demon-strated that collagen sheets can be used as the basis forcomposite bone tissue-engineering sca!olds [105]. Duobtained commercially available collagen sheets, precipi-tated HA onto the surface, then placed bone fragmentsalong the surface, rolling the composite into a tube. Thepore sizes in this material range from tens to hundreds ofmicrons; the material is absorbable and #exible. Cellsmigrated from the bone fragments into the matrix, sug-gesting that the material is bioactive.

4.2. Cellular systems: synthetic polymers

Polyglycolide (PG) "brous, nonwoven mesh is anothertissue-engineering candidate. Clinically well known andhaving the advantage of fast absorption, this material hasbeen applied to almost every area of tissue-engineeringresearch, including bone [106]. Polyglycolide mesh dem-onstrates relatively low mechanical integrity in vitro[107] and, for this reason, would be inappropriate asa bone tissue-engineering construct. By combining thismaterial with a second, reinforcing material, a stableconstruct can be formed. This has been accomplished inthe past using a PL solution to bond the mesh [108].More recently a speci"c bone application has been ac-complished by coating a PG-based tube with PL [106].Puelacher and coworkers applied PG mesh seeded withosteoprogenitor cells to the hollow portion of the stabil-ized tubes. These constructs showed promise as longbone defect replacements in a rat femoral defect model.

Since joints are the con#uence of several tissue types,including bone, researchers have recently undertaken thecomplicated task of reconstructing a whole joint. Thisrequires coculture, with each polymer section customdesigned to the speci"c cell type. Preliminary studies used


PL-stabilized PG mesh cocultured with chondrocytes,tenocytes, and periosteal osteoblasts in an attempt toform a "nger joint [109]. The separate parts were sewntogether with absorbable suture, and implanted subcu-taneously in nude mice. Although the eventual goal iscomplex, the basic concepts can be immediately appliedto osteochondral defect repair.

New synthetic polymers are also of great interest aspotential cellular sca!olds. Attawia and coworkers havefocused on poly-anhydride-co-imides as bone tissue-engineering sca!old alternatives [110]. These materialsabsorb by surface erosion, thus having the advantage ofa more predictable mass loss. Furthermore, they havehigh mechanical strength and rigidity, their compressivemoduli ranging from 10 to 60 MPa.

4.3. Cellular systems: ceramics

Solchaga and coworkers compared two commerciallyavailable HA-based constructs with a well-characterizedporous calcium phosphate ceramic [111]. Speci"cally,Hya! 11 (Fidia Advanced Biopolymers; Abano Terme,Italy) constructs (100}400 lm size pores, porosity of80%) were compared with ACP (Fidia Advanced Bio-polymers; Abano Terme, Italy) constructs (10}300 lmsize pores, 85% porosity). Porous calcium phosphateceramic with 60% HA, 40% TCP (200}400 lm poresizes, porosity of 60%) were also examined in this study.All materials were seeded with marrow progenitor cellsbefore subcutaneous implantation in rats. The ACP con-structs and TCP ceramics bound identical numbers ofcells, signi"cantly lower than those bound to the Hya!11. The ACP constructs absorbed relatively quickly with-out a protective cell layer, disappearing in the rat within3 weeks in subcutaneous location. The structure of Hya!11 is much more open with larger pores, allowing gooddistribution of cells throughout. The ACP constructstripled in volume when hydrated, perhaps diminishingthe available cell space within the pores. This demon-strated the advantage of high porosities in achievinggood cellular distribution and the importance of correctpore size selection [111].

5. Clinically available tissue-engineering options andspeci5cations

According to the food and drug administration (FDA),at this time there are no approved orthopaedic devicesthat incorporate tissue-derived components such as cellsand/or growth factors. Additionally, there is currently noconclusive data to support indications for these devices.There are, however, "llers without tissue-derived compo-nents that may be classi"ed as tissue-engineering mater-ials. They are listed in Table 2 along with manufacturername and FDA "ling classi"cation. The table lists both

premarket noti"cations (510(k)) and premarket ap-provals. Premarket noti"cations are from the manufac-turer to the FDA that state the intent of marketinga device for the "rst time or reintroduce a device to themarket that has been signi"cantly adapted. Premarketapproval is the most strict type of device application,requesting to take to market or continue marketinga Class III medical device. Included within the Class IIIFDA classi"cation are devices that `present a potential,unreasonable risk of illness or injurya and devices that`are of substantial importance in preventing impairmentof human healtha.

6. Future areas of development

Much work remains to be undertaken in the analysisof progenitor cells and their di!erentiation, particularlywith regard to their interactions with biomaterials. Bio-material development and "nal design will be essential tothe appropriate stimulation and di!erentiation of bonecells. The environment in which these polymer}tissuesystems are cultivated will greatly a!ect the long-termtissue viability. Furthermore, it will also be necessary tofocus on creating the optimal micromechanical environ-ment. Bioreactor studies examining the interaction ofcellular concentration with material type and the in#u-ence of residence time on bone development will also beof interest.

Clinically, it is always of interest to improve methodsof bone regeneration in order to reduce costs and thesurgical trauma to the patient. The ability to use auto-genous bone forming cells attached to a mechanicallysound, biologically active BMP impregnated, replaceablesca!olding would be ideal. This would allow implanta-tion of the cellular construct without associated surgeriesto harvest a graft or implant a structural device. Asa compromise, a similar cellular construct could be usedin conjunction with conventional "xation techniques.Both techniques would rely on a viable bed of host tissue,so that there is no local tissue necrosis or dense avascularscar tissue.

The ideal replacement material for osseous defectsassociated with orthopaedic reconstructive surgerywould be one that is initially pliable and readily moldedbut which hardens quickly once implanted. The photo-polymerizing polymers may "nd application in opensurgeries; they could also potentially be used in arthro-scopic applications or minimally invasive procedures.Polymeric foams may be good candidates as deliveryvehicles for cells or drugs such as antibiotics, since theywould conform to the defect.

Bone tissue engineering is one area that will bene"tgreatly from the current e!orts by the American Societyfor Testing and Materials to construct tissue-engineeringguidelines. It is very di$cult to correlate studies from


Table 2Current FDA-listed bone void "llers

Device type Product name Product description Regulation number Applicant

501(k) Devices, calciumsulfate bone void "llersor equivalent

Pro Osteon implant 500Rresorbable bone graftsubstitute

Calcium sulfate preformedpellets

K990131, cleared 3/2/99 Interpore Cross Interna-tional

Stimulan-calcium sulfatebone void "ller


K982663, cleared 2/26/99 Encore OrthopaedicsInternational

Pro Osteon implant 500Rresorbable bone graftsubstitute


K980817, cleared 9/25/98 Interpore International

Profusion bone void "ller Calcium sulfate preformedpellets

K973704, cleared 4/3/98 Biogeneration

Wright plaster of paris pellets Calcium sulfate preformedpellets

K963562, cleared 5/7/97 Wright Medical Techno-logy, Inc.

Wright plaster of parisbone void "ller kit



Wright plaster of parispellets (subject to revision)



510(k) Devices, Cranio-facial Bone Void Fillers

BSM * bone substitutematerial

Methyl methacrylate forcranioplasty

K983009, cleared 11/25/98 Etex Corporation

MEDPOR surgical granuleimplants


K982040, cleared 9/8/98 Porex Surgical, Inc.

Norian cranial repairsystem (CRS) bone cement


K973789, cleared 5/18/98 Norian Corporation

Bonesource hydroxyapatitecement (HAC)


K964537, cleared 1/24/97 Osteogenics, Inc.

Bonesource hydroxyapatitecement (HAC)


K953339, cleared 6/27/96 Osteogenics, Inc.

Cranioplastic, acryliccranioplasty material


K873689, cleared 10/26/87 Dentsply International

Premarket ApprovalDevices, Bone Fillersor equivalent

Collagraft Collagen matrix for bonerepair

P900039 Collagen Corporation

Pro Osteon 500 Porous hydroxyapatitebone graft substituteblocks and granules

P860005 Interpore CrossInternational

di!erent laboratories when each has an independent setof processing parameters or uses a speci"c polymer ofoften unreported physical parameters. Standardizationwill hopefully expedite the fabrication of successfultissue-engineered alternatives.

Acknowledgements

The authors wish to acknowledge the input of N.Y.Sloan and A. Torres-Cabassa from the United StatesFood and Drug Administration. The authors would alsolike to thank L. Jenkins of the Clemson University De-partment of Bioengineering as well as A.G. Mikos andJ.P. Fisher of the Rice University Department of Bio-engineering for their contributions.

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