Developed by the Federation of American Societies for Experimental Biology (FASEB) to educate the general public about the benefits of fundamental biomedical research.

INSIDEthis issueBBoonnee BBuuiillddeerrss::

TThhee SScciieennccee ooff GGrraaffttss��BBiioommaatteerriiaallss�� aanndd BBoonnee EEnnggiinneeeerriinngg

Bone Grafting: From Holes in the Head to Common Transplants

2From Coral to Glass:

Search for Bone Graft Substitutes

4Better Bone Repair Through Proteins

9Putting the Pieces Together:

Bone Grafts to Bone Engineering

10

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AcknowledgmentsBone Builders: The Science ofGrafts� Biomaterials� and Bone Engineering

Authors, Steven Stocker and Carrie D. Wolinetz, Ph.D

Scientific Advisor, Lynne Jones, Ph.D., Johns Hopkins

Medical Institutions

Scientific Reviewer, Paul Ducheyne, Ph.D., University of

Pennsylvania

BREAKTHROUGHS IN BIOSCIENCE COMMITTEEJames E. Barrett, Ph.D., Chair, Drexel University College of Medicine

Fred R. Naider, Ph.D., Past Chair, College of Staten Island,CUNY

David Brautigan, Ph.D., University of Virginia, School of Medicine

Marnie Halpern, Ph.D., Carnegie Institution of Washington

Tony T. Hugli, Ph.D., Torrey Pines Institute for

Molecular Studies

Richard G. Lynch, M.D., University of Iowa College

of Medicine

Mary Lou King, Ph.D., University of Miami School

of Medicine

Loraine Oman-Ganes, M.D., FRCP(C), CCMG, FACMG,

RBC Insurance, Toronto, Canada

BREAKTHROUGHS IN BIOSCIENCE PRODUCTION STAFF

Science Policy Committee Chair: Avrum Gotlieb, M.D., CM,

FRCPC, University of Toronto

Managing Editor: Carrie D. Wolinetz, Ph.D., Director of

Scientific Affairs and Public Relations, FASEB Office of

Public Affairs

Production Staff: Jennifer Pumphrey, Communications

Assistant, FASEB Office of Public Affairs

CCOOVVEERR:: The science of bone grafting and bone biomaterials

has provided extraordinary therapies for patients who have

suffered bone loss through traumatic injury� bone cancer� or

birth defects� In fact� bone and bone materials are the most

commonly transplanted materials after blood transfusions�

From late seventeenth century skull grafts to modern bone

bioengineering� scientists have taken their cues from funda�

mental physiology and even sea coral to develop replace�

ments for bone� Images from Science Photo Library�

iStockphoto� and the National Oceanic and Atmospheric

Administration�

1658_FASEB 7/7/09 9:10 AM Page C2

BUILDING BONE

Breakthroughs in Bioscience 1Breakthroughs in Bioscience 1

Bone Builders: The Science of Grafts�Biomaterials� and Bone Engineering

In the late seventeenth century,the Catholic Church threatened toexcommunicate a Russian noble-man named Butterlijin who hadrecently been treated for a skullwound obtained after a sword-fight with a Tartar soldier.According to a Dutch physician,Job Janszoon van Meekeren, whopublished a case study of theincident, the hole inflicted onButterlijin’s head was patchedwith a piece of the skull from adog by an anonymous and inven-tive surgeon. The merging ofhuman and animal tissue wasconsidered unacceptable to theChurch, but this first reportedbone grafting operation was aclear success: the wound hadhealed so well, physicians wereunable to remove the graft so thatthe hapless nobleman couldreturn to church. More than onehundred years later, in the 1820s,a surgeon named Phillips vonWalthers achieved similar excel-lent results when he repairedholes he had cut in patients’skulls to relieve pressure byreplacing the plugs he hadremoved (Figure 1). These werethe beginning steps towards solving a medical problem con-fronted by physicians even today:how do you replace missingpieces of bone?

According to the AmericanAcademy of OrthopaedicSurgeons, musculoskeletal dis-eases and conditions are amongthe most commonly reporteddebilitating illnesses, with nearly36 million Americans affected. In the intervening centuries sincevan Meekeren documented thecase of the wounded Russian,much progress has been made in perfecting bone grafts andfinding biologically compatiblematerials, or biomaterials, torepair diseased bone or to fill

bone voids caused by traumaticinjury, birth defects, or bone cancer surgery. Bone biomaterialsare also used to promote spinalfusion, in which bones in thespinal column are joined togetherthrough new bone growth in thespaces between existing bones.This is done to provide more stability to the spine or when adisc in between spinal bones isno longer viable. Breakthroughdiscoveries and decades ofresearch to understand how boneis formed, and what materials

FFiigguurree �� –– FFiirrsstt rreeccoorrddeedd bboonnee ggrraafftt:: The Phillips von Walther performed a successful allograft in ���� when� following surgery to relieveintercranial pressure byremoving a piece of skull� he repaired the hole with thepiece of bone removed fromthe patient’s own head� This sort of bone grafting surgery had been successfully performed over a thousandtimes by the early twentiethcentury� Source: ManchesterDaily Express/SSPL

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Breakthroughs in Bioscience 2

offer characteristics similar tobone tissue, have led to remark-able advancement in bone grafting and bone biomaterials,as well as forming the foundationof the next generation of cutting-edge bone engineering.

Bone Grafting: FromHoles in the Head toCommon TransplantsEvery year, more than 800,000

people in the United Statesreceive bone biomaterials, includ-ing bone grafts and bone graftsubstitutes. Bone is the mostcommonly transplanted tissueafter blood transfusions. Unlikemost tissues, bone is able toregenerate and reform withoutscarring, given the proper conditions and materials. (Seesidebar, “Basic Bone Biology”)Researchers and scientists havelearned to exploit this propertyover many centuries to create

modern day bone grafts and bonebiomaterials used as substitutesfor bone grafts.

A bone graft is bone that istransplanted from one area of theskeleton to another or from oneperson to another. Bone graftsobtained from other bones in thepatient’s own body, such as thecrest of the hip bone or the lowerpart of the thigh bone, are knownas autografts (Figure 2). Bone

Breakthroughs in Bioscience 2

grafts that come from a donor,such as a cadaver or someoneundergoing a hip replacement,are known as allografts. Whenthe bone material comes fromanother species, such as a pig ora cow, it is known as a xenograft.

The current gold standard inbone grafts is autografts. Thefirst autograft was that describedabove by von Walthers, and bythe 1920s over a thousand bone

FFiigguurree �� ––HHaarrvveessttiinngg ooff bboonneeffoorr aauuttooggrraaffttss:: Boneused in autografts is taken from thepatient’s own body�usually from thethigh bone or crestof the hip� Becausethe bone is beingharvested from thepatient� the size ofthe bone availablefor autografts is very limited� Figure designed byCorporate Press�

Basic Bone Biology

Bone is a type of rigid, connective tissuethat functions in support, movement,protection of vital organs, production ofblood cells, and storage of minerals. Boneconsists of inorganic materials, primarily a form of calcium called hydroxylapatite,and organic materials, including a proteincalled collagen and a variety of specializedbone cells. Osteoblasts are the main typeof bone-forming cell and they originatefrom stem cells that are found in the bonemarrow. Osteoblasts produce a complex,organic matrix called osteoid, which ismineralized or converted to a hard, inor-ganic substance, through deposits of calci-um. As the bone mineralizes around them,

osteoblasts become trapped in the bonematrix and are then known as osteocytes.Osteocytes no longer function to makebone, instead serving a communicationfunction, sending signals between differenttypes of bone cells.

Bone is constantly undergoing reformationor remodeling, through new growth and adestructive process called resorption. Inother words, as new bone is laid down,old bone is destroyed, creating a continualrebuilding of bone. As described above,osteoblasts are the main player involved ingrowth, but a different kind of cell, theosteoclast, is involved in resorption, which

prevents the new growth from significantlychanging the size and shape of the boneover time. The middle phase of remodelingis known as reversal, in which the resorp-tion of bone by osteoclasts stops and thebone is prepared by mononuclear cells forthe growth process involving osteoblasts.Remodeling of bone helps regulate the levels of calcium in the body (which isalternately stored and released duringgrowth and resorption), repairs microdam-age caused by everyday stress on thebones, and helps shape and support the skeleton during periods of growth.

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Breakthroughs in Bioscience 3

Making an Ideal Bone Graft:Osteoconductivity, Osteogenicity, and Osteoinductivity

What makes the perfect bone graft? There are three characteristics inherent to natural bone and autografts: osteoconductivity; osteogenicity; and osteoinductivity. The search for bone graft substitutes

has led scientists to try to find materials or combinations of materials that have all three properties.

The properties necessary for ideal bone grafts are somewhat analogous to those needed to grow plants.The osteoconductivity would be represented by the pot and the soil, the osteogenicity by the seeds,

and osteoinductivity by the fertilizer and water needed to encourage growth.

STRUCTURE – Osteoconductivity:the ability of a material to serve asscaffolding to support new bone formation and growth. Ideal osteoconductive materials are strong enough to provide support while alsohaving a porus structure throughwhich new blood vessels can formand new cells can migrate.

GROWTH – Osteoinductivity: the presence of growth factors, such as BMPs (protein structure shown), which recruit bone forming stem cells and encourage their transformation and growth into osteoblasts. Osteoinductive materials promote new bone formation and growth.

VITALITY – Osteogenicity: the presence of osteoblasts, a cell responsible for bone formation, or mesenchymal stem cells, which can transform into osteoblasts.Osteogenicity is the living, regenerative property of bone and bone grafts.

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autografts had been reported,most replicating von Walthersrepair of holes in the patient’shead. Because they come from apatient’s own body, there is lesschance of disease transmission orthe immune reactions experi-enced when transplanting foreigntissue. Autografts also promoterapid healing and successfulfusion with the existing bone.This is because autografts havethree important elements for newbone growth: osteoconductivity,osteogenicity, and osteoinductivi-ty. Osteoconductivity refers to theability of the graft to act as ascaffold with pores throughwhich blood vessels can growand fluids containing cells, proteins, and other substances for making new bone can enter.Osteogenicity (also calledosteogenic potential) refers to the presence in the graft of viable mesenchymal stem cells.Mesenchymal stem cells (MSCs)are a type of adult stem cellwhich have the capability to form many types of cell types,including bone cells, fat cells(adipocytes), cartilage (chondro-

cytes), muscle (myocytes), andfibroblasts. Under proper condi-tions, MSCs can mature into atype of bone forming cell calledosteoblasts (Figure 4). Specificbone growth factors can promotetransformation of the mesenchy-mal stem cells to osteoblasts.

Although bone autografts workwell, they are not without chal-lenges. Because doctors need toremove bone from the patientbefore they can transplant it,autografts involve an extra surgi-cal procedure with its associatedblood loss, postoperative pain,and possible complications, suchas injury to nearby nerves ormajor blood vessels. Manypatients experience pain at thesite from which the bone graft istaken for many months after sur-gery. Also, the amount of bonetissue that can be harvested islimited, particularly in olderpatients and infants.

Allografts, which use bonematerial donated by another per-son, solve many of the problemsrelated to autografts. Supply isnot as much of a problem with

allografts, because materials fromcadavers or living donors is read-ily available, although harvestingthe bone and testing it for possi-ble viruses, such as HIV and hepatitis B and C viruses, is cost-ly. However, bone allografts arenot without their own problems.In addition to the possibility of disease transmission, allograftsdon’t always fuse as well to exist-ing bone or heal as readily as doautografts, largely because of theway they are treated prior totransplantation. To preserve boneallograft material for future use,the bone is frozen or freeze-dried. These treatments usuallykill the cells that can form newbone. As a result, bone allograftsprovide primarily an osteocon-ductive matrix in which the bodyof the person receiving thegraft—the host—has to do all thework of forming new bone. Inother words, the allograft servesas a scaffold, but the stem cellsand growth factors needed togrow new bone may have tocome from the graft recipient orbe provided. For this reason, newbone takes longer to form in anallograft, and fractures may occurin the first year or two afterimplantation.

From Coral to Glass:The Search for BoneGraft SubstitutesThe limitations of autografts

and allografts have motivated scientists to develop alternatives,both by enhancing the ability ofallografts to fuse with existingbone and through development of bone graft substitutes through

Formation of Osteoblasts

FFiigguurree –– FFoorrmmaattiioonn ooff oosstteeoobbllaassttss:: Osteoblasts� the specialized cells critical to bonegrowth and reformation� are formed from mesenchymal stem cells found in bone marrow� These stem cells differentiate (become more specialized) into osteoblast precursors and then osteoblasts under the influence of growth factors� such as bone morphogenetic proteins or BMPs� Image by Corporate Press�

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FFiigguurree –– MMaarrsshhaallll UUrriisstt:: University ofCalifornia researcher� Marshall Urist�discovered a group of growth factorsknown as bone morphogenetic proteins(BMPs) while investigating abnormalcalcium deposits common in atherscle�rosis or hardening of the arteries� Innatural bone� BMPs stimulate bone cellgrowth� providing the osteoinductiveproperty of bone� BMPs are now usedclinically as part of the makeup of bonegraft substitutes to encourage bonegrowth and healing�

creation of biomaterials. Onesuch alternative, called deminer-alized bone matrix, is animprovement of the bone allograft. It was discovered byserendipity in the mid-1960s byMarshall Urist at the Universityof California, Los Angeles(Figure 4). Urist’s discovery wasable to explain an observationthat had long perplexed scien-tists: scrapings from healthy bone could help speed healing of fractures.

Urist had been trying to find themechanism by which abnormalcalcium deposits develop in certain diseases, such as athero-sclerosis, or hardening of thearteries. In one experiment, heexcised bones from laboratoryanimals and removed the calciumfrom the bones with mildhydrochloric acid, a processcalled decalcification. He thensoaked decalcified bone pieces indifferent concentrations of calci-um solutions, while leaving some

samples untreated. The purposeof the experiment was to seewhat effect varying the calciumlevel had on a part of the bodythat normally has lots of calci-um—namely, bone—in the hopeof identifying factors that causethe formation of abnormal calci-um deposits. (See sidebar, “Whyis calcium good for bones?”)

Urist implanted the decalcifiedand untreated samples in themuscles of rabbits and rats. Tohis surprise, new bone started

FFiigguurree �� –– DDeemmiinneerraalliizzeedd bboonneemmaattrriixx:: Demineralized bone matrix(DBM) is used as a bone graft substitute or extender for othertypes of bone graft substitutes�Made from ground bone� DBM is a form of allograft that can haveboth osteoconductive (structural)and osteoinductive (promotingbone growth) properties� It isavailable to surgeons in a varietyof forms� including as a powder�paste� or putty� so that it can bemolded to the shape necessary to replace the missing bone orbone fragment� Image courtesyof the Musculoskeletal TransplantFoundation�

“Why is calcium good for bones?”

Calcium is an important mineral in the

body, with functions ranging from heart

health to activating nerves and muscles.

It is also integral to the form and function

of healthy bones and teeth. Around seventy

percent of bone is made up of a calcium-

phosphate compound called hydroxylap-

atite. This mineral forms the hardened,

mineralized parts of the bone, needed

for strength and structure.

One of the functions of bone is to help

store calcium and release it when it’s need-

ed by other parts of the body, through the

process of bone remodeling. New bone

formation stores calcium, in the form of

hydroxylapatite, and resorption of bone

releases calcium into the bloodstream.

When you do not get enough calcium in

your diet, the body withdraws the calcium

it needs from where it is stored in the

bones. If you do not have adequate calci-

um to replace what has been removed,

over time this will weaken the bones and

could increase the risk of breaks, a condi-

tion called osteoporosis. (See FASEB

Breakthroughs in Bioscience article,

“Bone Builders: The Discoveries Behind

Preventing and Treating Osteoporosis.”)

Vitamin D is also important in bone health,

as it helps transport calcium to the bones.

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forming within a few weeks inthe untreated samples but tookmuch longer in the samples thathad been soaked in calcium solutions. Urist concluded thatdecalcifying, or demineralizing,bone uncovers a substance thatinduces rapid bone formationwhen implanted in a host. Helater called this substance bonemorphogenetic protein, or BMP.Subsequent work by Urist andothers found that there are actu-ally over 20 different bone morphogenetic proteins with different functions. Bone prepa-rations that removed the calciumto expose these BMPs becameknown as demineralized bonematrix.

Demineralized bone matrix(DBM) remained a researchmaterial until the early 1990s,when it was developed as a solu-tion to hospital power shortagesthat prevented the freezingrequired for preserving bone allografts. Demineralized bone ispulverized into a powder, whichpreserves the growth factors, likeBMPs, that allow it to promotebone growth. The powder is thenis mixed with additional materi-als to provide the optimal handling characteristics desiredby a surgeon. DBM is clinicallyavailable in gels, putties, pastes,and fabrics, all of which can be molded to fill the desired area(Figure 5).

Because demineralizing bonereleases BMP and other growthfactors, it has higher osteoinduc-tivity (stimulating good bone cellgrowth) than traditional allo-grafts. To increase its osteogenic-

ity (presence of bone-formingstem cells), DBM is sometimesmixed with bone marrow beforeuse. A recent study looking at theuse of bone graft options foundthat DBM was used in more than80 percent of trauma cases andhand/foot surgeries involvingbone grafts and it is often usedfor spinal fusion surgeries.However, because the matrix ismade through grinding into apowder, it does not provide thestructural support needed formany bone grafts. Because ofthis, DBM often does not fullysubstitute for a bone graft, butcan be used to extend the limitedmaterial available for a boneautograph or with other bonegraft substitutes, such as plasterof Paris.

Because of the limitations ofautografts, allografts, and DBM,scientists continued to search forbone graft substitutes: biomateri-

als that could act in place of natural bone. The first syntheticmaterial used for bone graftingwas bioabsorbable ceramics. Aceramic is a hard brittle materialproduced by firing nonmetallicminerals at high temperatures,while the term “bioabsorbable”refers to the fact that the ceramicis absorbed by the body as newbone is formed. The first bioab-sorbable ceramic used for bonegrafting was plaster of Paris.

Bioabsorbable ceramics aremade in such a way that theycontain interconnected pores, ascaffold-like system that allowsthe growth of blood vessels andthe influx of stem cells to formbone. The new bone forms at thejunction between the ceramic andexisting bone, often creating atight bond. This bond serves asan attractive place for local bonegrowth factors, such as bonemorphogenetic proteins (See

Ceramics were among the first mate-rials used to make artificial bone foruse as bone graft substitutes. Allceramics used as bone graft substi-tutes share the characteristics neces-sary for biomaterials used in anymedical application: they are non-toxic; elicit little to no immuneresponse; do not cause blood clots;are non-allergenic; and non-carcino-genic (do not cause cancer). The bioceramics currently used bysurgeons for bone grafting can bedivided into three main categories:bioabsorbable; bioactive; and bioin-ert. Bioasbsorbable ceramics will dissolve over time and are replacedwith new bone and/or surroundingtissue, whereas bioactive ceramicsform bonds with the existing bone.Bioinert materials have greaterstrength and are used in more com-plicated bone or joint replacements, such as for hip or knee prosthetics.One of the advantages of bioab-sorbable materials is that they can be used to carry doses of

growth factors, like the bone morphogenetic proteins (BMPs). As the material dissolves, the BMPsare released, helping the growth ofnew bone.

Two bioabsorbable ceramics commonly used as bone graft substitutes are tricalcium phosphate(TCP) and hydroxyapatite, a calcium-phosphate compound similar to thepredominant mineral found in naturalbone. Since TCP is absorbed as muchas 20 times faster than hydroxyap-atite, surgeons select TCP, hydroxya-patite, or combination of both basedon how fast they want the implant to be absorbed. If the implant isabsorbed too fast, bone may not havetime to form, while if the implant isabsorbed too slowly it may preventsome bone formation simply byoccupying space that should be occupied by bone.

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Breakthroughs in Bioscience 7Breakthroughs in Bioscience 7Breakthroughs in Bioscience 7

sidebar, “Ceramics: Not just forvases and pots”). Bioabsorbableceramics are readily available andcan be shaped to fit the piece ofbone that needs to be replaced(Figure 6). Unlike allografts,there is no concern over diseasetransmission, because these syn-thetic materials can be sterilized.They can also be engineered torelease bone growth factors,which helps new bone to grow.

Although bioabsorbable ceram-ics had clear advantages overautografts and allografts, they arenot without their own challenges.There is the chance that thepatient’s immune system mayreact badly to the foreign sub-stance. In addition, ceramicswere originally chosen as a bonegraft substitute because the porestructure was thought to be high-ly osteoconductive. Ideal bonegraft substitutes have intercon-nected pores, which allow bloodvessels to grow into the implant,thereby promoting new bonegrowth. However, there are limitsin the porosity (number of pores)in ceramic. While a large numberof pores were good for osteocon-

ductivity, the increase in the“holiness” of the ceramics makeit weak and prone to breaking, aless than ideal quality for a bonesubstitute. The limitations ofbioabsorbable ceramics led scien-tists and physicians to continuetheir hunt for a better bone graftsubstitute.

This focus on pore structure ledresearchers to an unexpectedsource: the coral found in tropicaloceans. Like Urist’s discovery ofbone morphogenetic proteins, thediscovery of coralline ceramicswas a case of serendipity. UnlikeUrist’s discovery, it involved sev-eral researchers in different fieldswho just happened to be at thesame university talking to eachother about their research. In the1960s, Jon Weber, a marine geol-ogist at the Pennsylvania StateUniversity (PSU), was studyingthe chemical composition ofcorals and other marine animalswith hard skeletons that he col-lected in the South Pacific. Whenhe found out that Eugene Whitein the university’s MaterialsResearch Lab was developinginstruments for characterizing

materials using scanning electronmicroscopy (SEM), he askedWhite if he could examine theskeletons of his marine animalsusing the microscope.

As a scuba diver, White firstbecame interested in coral whilediving in the South Pacific andnow found them equally excitingin the lab. When White looked atthe micrographs, he became fas-cinated by the uniform and inter-connected pores of some of theskeletons. “Having seen virtuallyevery type of man-made materialunder the SEM, it was obviousthat these marine animals weredoing something humans had notbeen able to do,” he wrote later ina paper in Materials ResearchInnovations. “Nature had provid-ed a whole range of architecturesof 3-D solids. These could be‘models’ or indeed templates.”

With no particular application in mind, White started makingmolds of the coral skeletons inceramics, polymers, and metals.In the summer of 1971, Whitewas joined in the lab by hisnephew, medical student Rodney

Breakthroughs in Bioscience 7Breakthroughs in Bioscience 7Breakthroughs in Bioscience 7Breakthroughs in Bioscience 7

FFiigguurree �� –– BBiioocceerraammiicc:: Bone bioceramic� for use in bone reconstruction� being treated with bone stem cells� The bioce�ramic is hydroxyapatite� a natural calcium phosphate mineral complex that is the crystalline component of bones and teeth�This synthetic bone mimics natural bone structure� Its porousstructure allows a type of precursor (stem) cell to grow anddevelop into new bone tissue� The bioceramic can be shapedinto implants that are treated with stem cells (obtained fromthe patient's bone marrow)� and grafted into the body toreplace missing bone or bone fragment� Source: Science Photo Library

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been developing from coral. Theorganization of pores in the coralwas very similar to that of naturalbone and had very high osteocon-ductivity (Figure 7).

One of the downsides of usingcoral skeletons as bone graft

Breakthroughs in Bioscience 8Breakthroughs in Bioscience 8Breakthroughs in Bioscience 8

White, who pointed out the simi-larity of some coral skeletons tothe porous ceramics and metalsthat were being developed forpromoting bone ingrowth. WhenRodney returned to medicalschool in Syracuse, NY that fall,he interested Richard Chiroff, anorthopedic surgeon, in conduct-ing bone implant studies in rab-bits using the substitutes he had

substitutes was that they weremade of calcium carbonate, achemical compound that wasquickly absorbed by the body,which didn’t allow for enoughtime for new bone to grow on the coralline scaffolding. Anotherscientist at PSU, Della Roy,developed a method of replacingthe calcium carbonate in the coralskeleton, which is absorbedquickly, with hydroxyapatite,which is absorbed more slowly

Breakthroughs in Bioscience 8

(Figure 8). This method is stillused for making a family ofcoralline ceramics. Othercoralline ceramics have beendeveloped as bone graft substi-tutes, containing mixtures ofhydroxypatite and calcium carbonate, which allow variationsin absorption times. Corallineceramics have the advantage ofbeing completely biocompatible,recognized by the patient’s bodyas so similar to bone that they do not cause a negative immunereaction.

As the scientists at PSU wereworking on coralline ceramics as

a bone graft substitute, a scientistat the University of Floridanamed Larry Hench was studyingthe electronic behavior of glass-ceramic semiconductors for the U.S. Department of Defense(Figure 9). He and his colleaguesdiscovered certain glass-ceramics

FFiigguurree �� –– DDeellllaa RRooyy:: A significantbreakthrough in the use or corallinebioaborbable ceramics as bone graftsubstitutes was made by scientist� DellaRoy� who developed a method of replac�ing the calcium carbonate in naturalcoral with hydroxyapatite� the materialfound in bone� Calcium carbonate wasabsorbed too quickly by the body� notallowing enough time for healing andbone growth to occur� making it ill suit�ed as a bone graft substitute� Source:American Ceramics Society

FFiigguurree –– CCoorraall ssttrruuccttuurree iiss ssiimmiillaarr ttootthhaatt ooff bboonnee:: As seen in these micro�graphs� scientists at Penn StateUniversity noticed that the structure of coral (image A) under the microscopelooked very similar to the structure ofnatural bone (image B) and the porousceramics that were being developed toact as bone graft substitutes� Ceramicsderived from sea corals are nowapproved for use as bone graft substi�tutes� Source: Science Photo Library and Dr� Rinard� Penn State University�

FFiigguurree �� –– LLaarrrryy HHeenncchh:: �� Illustratingthe serendipity that often drives scien�tific discovery� scientist Larry Henchdeveloped a bone graft substituteknown as Bioglass following a chanceconversation with a military officerabout the need for better biomaterials�Source: Science Museum/SSPL

A

B

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that conducted electricity evenwhen exposed to high radiationlevels. This meant that these newmaterials might be used as elec-trical switches in satellites wherethey could survive high energyradiation, such as that producedby solar flares or certain types of weapons.

In 1967, Hench shared a busride with a colonel who hadrecently returned to the U.S. aftera tour of duty with the ArmyMedical Corps in Vietnam. Thecolonel patiently listened asHench enthusiastically describedhis semiconductor experimentsand then asked, “If you can makea material that will survive expo-sure to high energy radiation, canyou make a material that will survive exposure to the humanbody?” He explained that thebody formed scar tissue aroundthe metallic and plastic parts thenavailable to surgeons. This oftenled to amputations, of which hehad witnessed many during histour of duty.

This got Hench thinking. Hesubmitted a proposal to the U.S.Army Medical Research andDevelopment Command to devel-op a material that would bond tobone without causing scar tissue.With the funding he received, hedeveloped a glass-ceramic thatlater became known as Bioglass®.Bioglass is made of a type ofglass that contains a high amountof calcium. Testing small rectan-gular Bioglass implants in ratthighbones showed that theimplants bonded firmly to thebone. “These ceramic implantswill not come out of the bone,”

BMPs Are Important for More Than Bone GraftsSince Urist’s initial discovery, bone morphogenetic proteins(BMPs) have since been identified in a wide range of invertebrateand vertebrate species, including worms, flies, and frogs. In each of these species, BMPs play key roles in embryonic development. For instance, the fly counterpart of the human BMP-2 and -4 genesis required for specifying which parts of the body wind up on topand which on the bottom. BMPs are also important in embryonicdevelopment of mammalian species—and not just for skeletaldevelopment. For example, BMP-7 is crucial for the developmentof the eyes and kidneys. Mutations in the genes that produce BMPsor their receptors are associated with a number of human diseasesand disorders affecting the skeleton and bone development.Fibrodysplasia ossificans progressive, or “stone man’s disease,” a rare and tragic disorder in which muscle and other soft tissue isgradually turned to bone, has recently been discovered to be causedby a mutation in a gene affecting BMPs. Further research on thesecritical proteins will help scientists develop treatments to this and other diseases.

remarked Ted Greenlee, who con-ducted the tests in the FloridaVeterans Administration Hospital.“I can push on them, I can shovethem, I can hit them, and they donot move.”

Subsequent research showedthat Bioglass is able to form sucha strong bond with bone becausea layer of hydroxyapatite formson the surface of the glass. Thehydroxyapatite binds to the pro-tein collagen, a key component ofbone. Although Bioglass forms astrong bond with bone, the mate-rial is brittle, which keeps it frombeing used in load-bearing situa-tions. It is currently used to makereplacements for bones in the

middle ear, which corrects a typeof hearing loss. It is also used tomake a stable ridge for denturesfollowing tooth extraction.

Better Bone RepairThrough ProteinsFrom the beginning, scientists

knew that the ideal bone graftsubstitute had to have the samecharacteristics as an autograft –osteoconductivity, osteogenicpotential, and osteoinductivity. In other words, an ideal bonesubstitute would have a porousstructure that would allow fusionwith existing bone and growth ofsupporting blood vessels whileholding up under load-bearing

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Breakthroughs in Bioscience 10

isolated about a millionth of anounce of protein that caused boneto form when implanted underthe skin of rats. To their surprise,the scientists discovered thatwhat they thought was a singleprotein was actually a family ofrelated proteins. The informationthey were able to gather fromstudying this family of proteinsallowed the Genetic Instituteresearchers to use genetic engi-neering technology to synthesizehuman BMP-1 through -7.Finally, scientists held the key tothe factors critical for encourag-ing bone to grow.

BMPs regulate bone formationby recruiting mesenchymal(bone-forming) stem cells to theplace where new bone is needed,stimulating them to grow andreproduce, and then causing themto transform into mature bonecells called osteoblasts.Interestingly, only one type ofBMP is needed for forming bone.Providing two or more at thesame time does not have an additive effect. However, admin-istering a higher dose of a BMPcauses bone to form sooner andin greater amounts.

Besides being important inskeletal development in theembryo, BMPs stimulate boneformation during fracture repair.Two BMPs are currently availablefor clinical applications: humanBMP-2 and -7. To keep theBMPs where they are needed, the proteins are added to a carri-er, such as a collagen sponge thatis absorbed by the body as newbone is formed. A large numberof animal studies have shown that

both BMPs can be used to healfractures and fuse spinal bones,often with results that were betterthan those obtained with boneautografts.

Currently, the Food and DrugAdministration has approved theuse of human BMP-2 for helpingto treat fractures of the tibia, orshinbone, and for helping to fuse spinal bones in the lowerback. Human BMP-7 has beenapproved for treating fractures oflong bones in cases in which thefractures show no sign of healing,use of autograft is unfeasible, andalternative treatments have failed.It has also been approved for use in fusing spinal bones in the lower back for patients inwhom standard treatments areunfeasible.

Putting the piecestogether: From BoneGrafts to BoneEngineeringWhen bone is damaged, the nat-

ural repair process involves theformation of a porous mineral-ized matrix, the growth of bloodvessels through the pores, and theinflux of cells to form bone onthe surface of the matrix. Thewhole process is directed by sig-naling molecules, such as theBMPs. To repair large bonedefects that will not heal ordinar-ily, scientists are trying a varietyof approaches, but they all mimicthe natural repair process. Assuch, they usually involve aporous mineralized matrix orscaffold to which is added bone-forming cells, signalingmolecules, or both. Putting

pressure, the stem cells needed toregenerate the bone tissue itself,and growth factors to promotegrowth of the new bone tissue.

Neither DBM nor the syntheticbone graft substitutes met all ofthese requirements. For instance,none of them provide muchstrength and, as such, they aremost appropriate for filling smallholes in bone. When used to fill a large area of missing bone, sur-geons must support the sectionwith a variety of hardware untilnew bone is formed. Also, thesematerials provide little more thanan osteoconductive matrix. DBMhas some osteoinductive capacity,but it pales in comparison tobone autograft and growth factors may be reduced as it isprocessed. The ceramics andglass could provide the structure(osteoconductivity) and bonemarrow could provide the stemcells (osteogenic potential), butresearchers still needed to find away to increase bone graft substi-tute’s ability to promote regenera-tion of bone tissue: an increase inosteoinductivity.

The need for a better alternativeled scientists to return to Urist’sbreakthrough discovery of bonemorphogenetic protein (BMP).Twenty years were spent trying topurify what was then thought tobe a single protein. Finally, abreakthrough came in 1988 withtwo studies by John Wozney and colleagues at the GeneticsInstitute, a biotechnology compa-ny in Cambridge, Massachusetts.

Starting with nearly 90 poundsof bone powder from cows, they

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Breakthroughs in Bioscience 11Breakthroughs in Bioscience 11Breakthroughs in Bioscience 11

together the pieces of break-through discoveries describedabove forms the foundation of thefield known as tissue engineeringof bone, or simply bone engineer-ing. This is the cutting edge ofcreating replacement bone and an active area of research.

For scaffolds, many scientistsutilize the same materials that are used as bone graft substitutes,including demineralized bone

Breakthroughs in Bioscience 11

FFiigguurree ���� –– BBoonnee eennggiinneeeerriinngg:: The cutting edge of bone replacement is known as bone engineering� which puts the knowledge gainedthrough years of studying bone biology and bone grafts to create new bones for transplant� Patrick Warnke� a German scientist� madean exciting breakthrough in bone engineering when he was able to replace a patient’s missing jaw bone or mandible with new bone� As shown in image A� a wire mold was built using a computer�aided design of the patient’s skull� The mold was filled with bone mineraltaken from cows� growth factors� and stem cells from the patient’s bone marrow (image B)� For the next weeks� the mold wasallowed to incubate inside the patient’s body� having been surgically implanted in the armpit� an area rich with blood vessels (image C)�The new bone was removed and implanted in the patient’s jaw allowing him to eat solid food for the first time in more than � years�From Warnke et al� (���) The Lancet� �(��): ��� �� Used with permission�

A

B

C

matrix and the bioabsorbableceramics. Also used are syntheticpolymers, a polymer being alarge molecule consisting of alinked series of repeating units,or monomers. The polymers usedin bone engineering degrade inthe body into carbon dioxide andwater. Polymers are attractivechemicals for constructing scaf-folds because bone growth factors can be incorporated into

the polymers and then released at a steady rate as the polymerdegrades.

The cells used in these experi-ments are mesenchymal or boneforming stem cells, usually iso-lated from the bone marrowfound in the cavities insidebones. From a small amount of bone marrow—10 to 20 milliliters—stem cells can be iso-lated and expanded in cell culture

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Breakthroughs in Bioscience 12

into billions of cells. They canalso be frozen and preservedwithout any loss of ability toform new bone cells. Instead ofpure, isolated stem cells, someresearchers just use bone marrowiteself, which in addition to stem cells also con-tains bone growth factors andblood-forming cells.

An important question in bone engineering is whether it is more effective to use cells, bonegrowth factors, or both in con-structs to heal bone defects.Using sheep, Frank den Boer and colleagues at VU UniversityMedical Centre in theNetherlands compared BMP-7with bone marrow for repairingbone and found that both pro-duced about the same results.However, several studies haveshown that combining bonegrowth factors with bone-formingcells yield better results thanusing either by themselves.

In these studies, as in most ofthe research discussed so far, scientists are using natural orsynthetic materials to fill holes in long bones. The holes are toobig to heal on their own, but theyhave a simple shape. In clinicalpractice, patients are often inneed of large sections of boneswith complicated shapes. Forinstance, a patient undergoing hip replacement therapy is inneed of a new femoral head,which is shaped like a ball to fitinto the socket of the hip bone.

Currently, surgeons replace thefemoral head with a ball made of metal or a hard ceramic that is not absorbed by the body.

This unit is attached to a longmetal stem that is inserted intothe hollow canal inside the femur for support. Although thisarrangement has worked well for many patients, replacing theentire top part of the femur withnew bone may work better.

A bone engineering experimentconducted by Roger Khouri and colleagues at WashingtonUniversity suggests that this idea may not be that far-fetched. The scientists created molds outof silicone in shapes of various bone parts. Into the molds theresearchers placed muscles fromrats, which served as the sourceof stem cells, similar to thosefound in bone marrow. The scien-tists also added demineralizedbone matrix and one of the BMPsto promote bone growth. Finally,they placed the filled molds intothe bellies of rats to incubate.After 10 days, the molds wereremoved and opened. Inside themolds they found well-definednew bone in the shape of themolds. Khouri and his colleagueshad put together the pieces fromdecades of fundamental discoveryto create new bone and a promis-ing method for repairing large,bony defects.

Using a similar technique,Patrick Warnke and colleagues at the University of Kiel inGermany were able to replace alarge section of a patient’s lowerjawbone, or mandible, with newbone. The patient had had abouttwo-thirds of his mandibleremoved due to a bone tumoreight years previously, and themissing bone had been replaced

with a metal plate. He was onlyable to eat soft food and soup andhad trouble pronouncing words.Due to his problems eating andspeaking, he had isolated himselfsocially and was feelingdepressed and suicidal.

Based on a three-dimensionalscan of the patient’s head,Warnke’s team designed a virtualreplacement of the missing boneusing a computer program. Basedon the design, a Teflon modelwas created and used as a moldto form a titanium mesh scaffold.After removing the model, thescaffold was filled with bonemineral, BMP-7 in a collagencarrier, and the patient’s bonemarrow. The titanium mesh scaffold with its cargo was thensurgically implanted under amuscle in the patient’s rightarmpit, an area rich in blood vessels (Figure 10).

After letting the ingredientsincubate in the body for 7 weeks,the researchers removed the scaffold and the bone that had formed inside, along with anadjoining part of the muscle andthe local artery and vein that hadgrown into the implant. The sur-geons removed the metal platethat had been taking the place of the missing jaw bone andreplaced it with the bone-muscle-blood vessel implant. Usingmicrosurgical techniques, theyconnected the blood vessels inthe implant to blood vessels inthe head so that the new bonewould continue to have a bloodsupply.

By the 4th week post-transplan-tation, the patient was able to eat

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Breakthroughs in Bioscience 13

solid food for the first time innine years. He was also able tospeak more clearly and resumedsocializing with family andfriends. His mood improved and he gained weight.

Bone engineering in humans isstill in the experimental stage andthere is a great deal of work to be done. Even so, remarkableprogress has been made since ven Meekeren reported on theRussian soldier and his dog skullpatch. From the day that MarshallUrist first implanted demineral-ized bone matrix in the musclesof laboratory animals and sawbone form to White’s firstglimpse of coral’s bone-likeskeleton, incremental discoveriesby scientists and physicians havebrought us to the brink of creat-ing new bone for transplant.

BiographiesSteven Stocker writes about biomedical research and health from the Philadelphiaregion. She has written for Discover, Glamour, Physician’s Weekly, Consumer Reports onHealth, The Washington Post, Los Angeles Times, Dallas Morning News and numerous otherpublications. She also writes frequently for the National Institutes of Health and the NationalAcademy of Sciences. This is her seventh article in the Breakthroughs in Bioscience series.

Carrie D. Wolinetz, Ph.D., is the Director of Scientific Affairs and Public Relationswithin the Office of Public Affairs at the Federation of American Societies for ExperimentalBiology (FASEB). Dr. Wolinetz works on a portfolio of issues on behalf of FASEB, includ-ing the use of animals in research, cloning and stem cells, biosecurity, and federal fundingof research. She has a B.Sc. in animal science from Cornell University and received her doc-torate in animal science from the Pennsylvania State University, where her area of researchwas reproductive (oviductal and gamete) physiology.

Lynne Jones, Ph.D., is an Associate Professor and Director of the Center forOsteonecrosis Research and Education and Arthritis Surgery Bone Bank at the JohnsHopkins Medical Institutions. Her research interests include osteonecrosis pathogenesis andtreatment, total joint arthroplasty successes and failures, evaluation of bone graft materials,and animal models of musculoskeletal disease. Dr. Jones currently serves as the President ofthe Society for Biomaterials and has also been selected as a Fellow, Biomaterials Scienceand Engineering, but the International Union of Societies for Biomaterials and Engineeringfor her work in the field. In addition, Dr. Jones serves as a Member-at-Large for theOrthopaedic Research Society.

We are almost at the point where an oft-quoted childhood rhymemight be transformed to “Sticksand stones can break your bones,but scientists can replacethem…”

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