novel approaches to fracture healing

$: Novel approaches to fracture healing$
Niyibizi & KimNovel approaches to fracture healing Novel approaches to fracture healing

Christopher Niyibizi & Myung Kim

University of Pittsburgh School of Medicine, Musculoskeletal ResearchCenter, Department of Orthopaedic Surgery, 200 Lothrop Street, RM C313,Pittsburgh, PA 15213, USA

The fractures that occur as a result of trauma frequently require multiplestage surgical procedures to achieve adequate union. Bone grafting withautogenous cancellous or cortico cancellous bone grafts is the traditionalmethod used to repair bone defects. Most fractures will heal using thistraditional procedure, however a number of fractures, up to 10% of thecases in United States alone, will result in delayed or impaired healing.Novel approaches are currently being investigated for the augmentationand acceleration of fracture healing. Some of these approaches include theuse of biodegradable matrices; cell based approaches supplemented withosteogenic factors and genetic therapy. Cell based approaches for fracturehealing have roused intense interest because of the great advance in theisolation and expansion of cells from the marrow that have the ability todifferentiate into various types of cells including osteoblasts. In addition,the discovery and cloning of several proteins (bone morphogeneticproteins) that have the ability to induce bone formation, have contributedto the investigation of novel approaches to augment fracture healing. Use ofgenetic therapy for the augmentation of fracture healing has also recentlygained strong interest. The attractive feature of gene therapy is thattherapeutic proteins can be delivered locally to the fracture site in relativelyhigh concentrations and in a sustained fashion. This review discusses thesenovel approaches and presents an assessment of their future clinicalapplicability.

Keywords:BMPs, bone healing, gene therapy, osteoprogenitor cells, scaffolds

Exp. Opin. Invest. Drugs (2000)9(7):1573-1580

1. Introduction

It is estimated that 5.6 million fractures occur annually in the United Statesand about 5 - 10% of these result in delayed or impaired healing [1]. Thesegmental bone defects occurring as a result of trauma, frequently requiremultiple stage surgical procedures to obtain adequate union. Thetraditional method used to repair segmental bone defects is bone graftingwith autogenous cancellous or cortico cancellous bone grafts. Althoughmost of the fractures will heal using this procedure, up to 15% of cases needmultiple stage surgeries to reach bone union. The availability of autogenousbone graft is limited to a few donor sites in the skeletal system. When theautogenous graft is harvested from the iliac wing, there is surgical morbidityassociated with the harvesting procedure, such as donor site pain, paresthe-sias and infection [2-5]. Use of allograft bone alleviates some of theseproblems but there is a potential risk for bacterial infection, immunogen-icity and transmission of systemic diseases such as HIV and hepatitis [6,7].As a result of these potential problems, novel approaches are being investi-gated for the augmentation of fracture healing. In this review we will

15732000 © Ashley Publications Ltd. ISSN 1354-3784

Review

1. Introduction

2. Scaffolds and fracturehealing

3. Cell-based approaches tofracture healing

3.1 Bone marrow cellsenriched inosteoprogenitor cells

4. Growth factors andfracture healing

5. Genetic therapy andfracture healing

5.1 Gene delivery to thefracture site

6. Expert opinion

7. Conclusions

Bibliography

http://www.ashley-pub.com

Expert Opinion on Investigational Drugs

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discuss some of the approaches that are being investi-gated for the augmentation of fracture healing andtheir potential clinical application for humantreatment.

2. Scaffolds and fracture healing

A number of investigators have shown that demineral-ised bone matrix can serve as a bone-inducing agent(osteoinductive) and/or as a scaffold (osteoconduc-tive) to repair skeletal defects [8,9]. Consistent healingof bone defects has been achieved using a combina-tion of bone marrow and demineralised bone matrix[10]. The osteogenic effect of demineralised bonematrix was attributed to its osteoconductive proper-ties rather than to its osteoinductive or stimulatoryproperties. However, mineralised bone matrix is anon-viable foreign material in the fracture defect andthus could lead to resorption lacunae in the newlyforming bone and to early infection. In addition todemineralised bone matrix, investigators are seekingother biomaterials that are biocompatible, biodegrad-able and osteoconductive. These biomaterials wouldsupport mesenchymal stem cells and create anenvironment for the cells to differentiate intochondrocytes and osteoblasts. Synthetic biomaterialshave been shown to deliver a high density of autolo-gous cells into the host for the formation of newfunctional tissue [11]. Some of the synthetic polymersthat have been tried include polyglycolic acid, whichhas been shown to degrade into non-toxic products[12,13]. However, these polymers tend to generatelarge amounts of lactic or glycolic acid in vivo andcould lead to the demineralisation of bone [14]. Othermaterials based on calcium phosphate have and arebeing evaluated for clinical use [1,15,16]. For examplea hydroxyapatite-based biomaterial that is formed bythe conversion of a marine coral calcium phosphate tocrystalline hydroxyapatite has been approved for usein augmenting bone repair [19]. Clinical trials usingthis biomaterial showed efficient healing of fracturesas well as bone defects [17,18]. Use of resorbablebiomaterials in combination with cells that areenriched in osteoprogenitor cells may offer greatbenefit in augmenting fracture healing.

3. Cell-based approaches to fracture healing

Alternative approaches to bone grafting haveincluded the use of autologous bone marrow toaugment bone healing. This is based on the

hypothesis that bone marrow contains osteogenicprecursors that could contribute to the formation ofbone [20-23]. Autogenous bone marrow have beenused clinically to augment the osteogenic response toimplanted allografts and xenogeneic bone grafts[24-26]. A bone graft substitute has to be both osteoin-ductive as well as osteoconductive. Osteoinductivematerials actively stimulate new bone formation byinducing mesenchymal stem cells to differentiatetowards an osteoblastic lineage. Osteoconductivematerials provide a matrix or scaffold on which newbone may be deposited.

Bone marrow alone has been used clinically as anosteogenic graft by some investigators [27]. Studies inanimal models and humans have demonstrated theeffectiveness of bone marrow to heal large bonedefects [28-30]. A problem observed in animal studiesand clinical cases of non-unions treated with marrowinjections, is the tendency of the injected marrow todiffuse away from the fracture site. The bone marrowinjections however, were able to induce fracturehealing but they did not accelerate the healingprocess.

3.1Bone marrow cells enriched inosteoprogenitor cells

The number of cells in the whole marrow that havethe potential to differentiate into chondrocytes orosteoblasts is very small. The major effort is nowaimed at using bone marrow cells that are enrichedwith cells that have the potential to give rise tochondrocytes and osteoblasts. Cells that have thepotential to give rise to a variety of different cells havebeen isolated from the marrow of different animalspecies [31,32]. The isolated cells have been shown togive rise to not only osteoblasts but also chondro-cytes, fibroblasts, myocytes and adipocytes [23,32,33].These cells have been named mesenchymal stem cellsby some investigators and others refer to these cells asbone marrow stromal cells [22]. The cells have beenshown to retain multipotency even after severalpassages [33]. Clinical applicability of these cells hasbeen demonstrated in animal studies using critical sizedefects as well as segmental defects [34]. Thecombination of mesenchymal stem cells with asuitable scaffold may offer a more superior andaccelerated healing process than whole bone marrowalone.

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Invest. Drugs(2000)9(7)

1574 Novel approaches to fracture healing

4. Growth factors and fracture healing

In addition to supplying the defect site withosteogenic cells and a structural carrier, investigatorsare also examining the use of selected growth factorseither in combination with osteoprogenitor cells oralone. Of all growth factors, the most osteoinductive isa group of proteins known as the bone morphoge-netic proteins (BMPs) [35-38]. These proteins aremembers of the transforming growth factor (TGF)-βsuperfamily, a large family of secreted signallingmolecules [37]. Several members of this gene familyhave been identified [37,38]. Investigation into the useof these proteins in fracture healing is presently amajor goal of many investigators. The major functionof BMPs is to induce new bone formation. Numerousstudies have demonstrated that BMPs can efficientlyheal large bony defects as well as segmental defects.The proteins that have received much of the attentionare BMP-2 and BMP-7 (or osteogenic protein-1).Recombinant proteins have now been generated andare currently undergoing clinical trials. BMP-2, whencombined with inactivated demineralised bone matrixused as a carrier, has been shown to induce de novocartilage and bone formation in rat, sheep and dogbone defect models [39-41]. BMP-7 has also beenshown to heal large segmental defects in animalmodels [42]. These BMPs show great promise inaccelerating and healing bone defects and fractures.Most of the studies on the use of BMPs have relied onthe delivery of these factors in protein form. Thehalf-life of these proteins is very short and thereforesustained release of these proteins is inefficient, thuslarge amounts of the protein are needed to repairfractures. Suitable carriers for the slow release ofBMPs in fractures are currently under intense investi-gation [43]. Other growth factors that are beinginvestigated include vascular endothelial growthfactor (VEGF), this factor has been shown to stimulateosteoblastic precursor cells to differentiate intoosteoblasts [44]. The vascular system is essential forbone healing in providing oxygen and nutrients,whilst also removing metabolic waste products. Oncethe local blood flow or the microcirculation isdisturbed, even non-complicated fractures end up asnon-unions. Angiogenesis, the sprouting of newcapillaries, plays an important role in the healing ofdamaged tissues, including bones. VEGF was found topromote angiogenesis as part of co-ordinated tissuerepair. Exogenous VEGF was found to induce newblood vessel formation and to increase perfusion inischaemic rabbit limbs. Endothelial cells throughout

the vascular system can respond mitogenically toVEGF [44,45]. In addition, VEGF has been shown toupregulate BMP-2 expression.

LIM mineralisation protein (LIMP-1) is an osteoinduc-tive factor that has recently been shown to be effectivein the augmentation of spine fusion in athymic rats[55]. This osteoinductive factor is a transcription factorthat was discovered by differential displaypolymerase chain reaction after treating osteoblasticcells with glucocorticoid [56]. LIMP-1 expression wasshown to be regulated by BMP-6 [57]. As LIMP-1 is atranscription factor, it can only have an effect if it isdelivered into cells. This osteoinductive factor holdsgreat potential for use in the augmentation of fracturehealing when it is delivered by gene transfer (seebelow).

5. Genetic therapy and fracture healing

As discussed above, there is strong evidence thatosteogenesis and fracture healing can be augmentedby exposure of osteoprogenitor cells to growth factorssuch as BMP-2, or BMP-7, LIMP-1 and, possibly,VEGF. One problem in using these factors clinicallylies with their delivery to the sites of interest in asustained fashion. The biological half-lives of thesemolecules is in the order of minutes to hours, whereasbone healing occurs over a period of weeks tomonths. It is impractical to re-administer growthfactors to sites of segmental defects on a sufficientlyfrequent basis, thus several investigators are nowexamining the potential of using gene therapy as atool to deliver growth factors of interest to the fracturesites. The aim is to use a technology that wouldmaintain high local concentrations of growth factorsfor extended periods of time. In this context, genetransfer of growth factor genes into fractures, toaugment fracture healing, is actively beinginvestigated.

5.1Gene delivery to the fracture site

There are two methods by which a gene encoding aspecific growth factor can be delivered to the fracturesite. One is the ex vivo method and the other is the invivo procedure [46,47]. The ex vivo method involvesgenetic modification of mesenchymal stem cells byintroducing growth factor genes into the cells thatwould augment fracture healing. The geneticallymodified cells are then implanted into the fracture siteusing a suitable scaffolding material. In this situation,


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the implanted cells act as sources of the therapeuticproteins. The in vivo technique involves directinjection of the vector encoding the therapeuticprotein into the fracture site. These two approachesare discussed below.

5.2Ex vivogene transfer

As discussed above, bone marrow stromal cells arecurrently being investigated as vehicles for ex vivogene transfer [33,48,49]. Transduction of osteogeniccells with genes encoding BMP-2, BMP-7, LIMP-1 oreven VEGF endows these cells with the ability tosynthesise these growth factors in large amounts.When the transduced cells are returned to the fracturesite they serve as endogenous sources of these factorswhich, by autocrine and paracrine mechanisms,induce the formation of bone and angiogenesis.Vectors based upon retroviruses and adenoviruses arecurrently being evaluated [47]. Retroviruses whichintegrate into the host cells’ chromosomal DNA, offerthe prospect of long-term gene expression. It hasbeen shown that retrovirally transduced stromal cells,when infused into mice femurs, continue to expresstheir transgenes in bone marrow in vivo for anextended time period [33]. The disadvantage of theretrovirus is that they require dividing cells. Normally,osteoprogenitor cells in vivo undergo very slowdivision. Adenoviruses do not generally give suchprolonged gene expression, but they are highlyinfectious and extremely high levels of gene expres-sion can be achieved. These vectors are currentlybeing investigated as the preferred choice for thetransduction of bone marrow stromal cells [50-51].

The applicability of this technology has beendemonstrated in rat and rabbit fracture healingmodels [51,52]. A segmental defect was created in a ratmodel and mesenchymal stem cells, transduced withan adenovirus encoding BMP-2, were delivered to thefracture site in a demineralised bone matrix. Eightweeks after the implantation of osteogenic cells(transduced with the BMP-2 gene) into the segmentaldefects, which would normally not heal, the investiga-tors demonstrated bone formation and completeunion in 22 out of 24 experimental animals [51]. Thesegmental defects that received osteoprogenitor cellsalone, or had no cell transduction, did not heal, or insome animals, achieved a poor level of healing [51].These findings indicated that gene therapy might be auseful tool for the delivery of osteogenic factors to thefracture site. Another ex vivo approach using the novelLIMP-1 has been tested in the athymic rat model of

spine fusion [55]. In this study, the investigatorsdemonstrated that bone marrow cells transduced witha cDNA encoding the LIMP-1 gene induced spinefusion in the athymic rat model [55]. The attractivefeature of the LIMP-1 gene is that only very few cellsneed to be infected and persistent gene expression isnot necessary. The hypothesis is that this transcriptionfactor induces a cascade of events that culminates inthe induction of several other osteoinductive factorswhich initiate bone formation [55].

5.3 In vivo gene transfer to fracture site

In vivo gene therapy involves direct delivery of thevectors encoding therapeutic genes into tissue ororgans. The applicability of this procedure wasdemonstrated in a rabbit model of fracture healing[52]. The investigators created a femoral segmentaldefect in a rabbit. The fracture site was injected withan adenovirus expressing either a Lac-Z gene thatencodes the β-galactosidase, or an enzyme luciferase.The presence of Lac-Z positive cells was determinedby histological analysis of the tissue sectionsharvested from the injected site, surrounding boneends of the injected site and other surrounding tissues.Luciferase activity was analysed by enzyme assays[49,52]. The investigators demonstrated that directinjection of the virus resulted in the transduction ofthe cells in the cut ends of the bone, scar tissue and thesurrounding muscle. These investigators alsodemonstrated that the gene expression persisted up tosix weeks in bone, while it was lost between two andsix weeks in muscle and the scar tissue. Using thesame rabbit model of fracture healing, the investiga-tors used a therapeutic gene to assess the feasibility ofthis approach to heal segmental bone defects. Theauthors clearly demonstrated that the injection of anadenovirus containing BMP-2 cDNA into the fracturesites created in the femurs of rabbits, induced healingof the fractures by eight weeks after BMP-2 genedelivery. The defects that did not receive the gene didnot heal [58]. Biomechanical testing of the rabbitfemurs twelve weeks after injection of the viruscontaining the BMP-2 gene into the fractures,confirmed that these femurs were biomechanicallysuperior to the femurs that did not receive the BMP-2gene [58].

A different approach of in vivo gene transfer toenhance fracture healing involves the use of expres-sion plasmids containing the DNA of interest. In thisapproach, the investigators have developed atechnique in which the plasmid DNA that encodes a



protein of interest is incorporated into a threedimensional structural matrix [53,54]. The compositeof the DNA and the matrix, which has been termedgene activated matrix, can be delivered locally to thefracture site. The activated matrix acts as an osteocon-ductive material in which mesenchymal stem cellsmigrate and come into contact with the plasmid DNAand then take up the DNA. The cells that take up theDNA express the gene by synthesising the desiredtherapeutic proteins. The applicability of this conceptwas demonstrated in a rat model of fracture healing[53]. In this study the investigators created a criticaldefect in a rat femur that would normally not heal butwould result in a non-union. Application of the geneactivated matrix containing the plasmid DNAencoding BMP-4 or parathyroid hormone (hPT1-34)demonstrated induction of healing by the plasmidDNA expressing the therapeutic proteins in both therat model and also in a dog model [54]. These datademonstrated that it is possible to apply in vivo genedelivery to augment fracture healing.

6. Expert opinion

The use of locally administered growth factors toaccelerate fracture healing is currently an active areaof investigation [51]. BMPs have been shown topossess the potential to promote the formation of newbone in different animal models. VEGF has beenshown to improve the microcirculation, essential forbone healing, by stimulating local angiogenesis.These new approaches induce local therapeutic levelsof these growth factors and will lead to a developmentof novel minimal invasive techniques to influence therepair and regeneration of bone even after maximaltraumatic injuries. Genetic therapy offers much morepotential for the augmentation of fracture healing,however there exist some hurdles that need to beovercome before this technology can be appliedclinically. Although adenoviral vectors efficientlytransduce all types of cells and give high gene expres-sion, they can also lead to inflammation due toimmunological response. When vectors that are safeand can deliver genes to cells and to fracture sitesefficiently are developed, this approach ofaugmenting fracture healing holds great promise. Theactivated gene matrix shows great potential as anattractive procedure to accelerate and to augmentfracture healing. The attractive feature of thisapproach is that it avoids the use of viral vectors,which, as discussed above can lead to an inflamma-tory response. The drawback of this approach is thatin general, non-viral vectors do not give high geneexpression. However, low doses of therapeuticproteins may be sufficient so that the low level geneexpression of the therapeutic proteins may beadequate to induce fracture healing. These novelapproaches to fracture healing, in combination withthe knowledge that has been accumulated regardingthe BMPs, hold great promise in the acceleration andaugmentation of fracture healing. These approachesare especially attractive for the augmentation of thehealing of bone non-unions. Non-unions usuallyresult from poor vascularity and the formation of thefibrous tissue separating the defect. Use of osteogeniccells that are expressing, for example BMP-2 gene,would serve as repair cells at the same time also asgenerators of growth factors that would induceangiogenesis and chemotaxis of the reparative cells.Although these approaches, especially genetictherapy, appear promising, a great deal of workremains to be done before this technology can be


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Bone marrowmesenchymalstem cells

Virusencodingosteogenicprotein

In vivo Ex vivo

Plasmid DNA orvirus

Scaffold

Figure 1: Schematic drawing illustrating two procedures forintroducing genes into fracture sites.In vivo, involves directinjection of vectors encoding osteogenic factors into thefracture site.Ex vivo, involves injection of mesenchymal stemcells transduced with vectors encoding osteogenic factors.Modified from [49].

applied for human treatment. The duration andamount of therapeutic growth factor proteins neededto heal the fracture has to be determined. In addition,the side effects of the osteogenic proteins producedby the transduced cells and the cells themselves arenot known. Whether overproduction of theosteogenic growth factors could lead to undesirableeffects remains to be determined.

7. Conclusions

The novel approaches to fracture healing discussed inthis review hold great promise for the future oftreating fractures once the mentioned obstacles areresolved. Use of osteogenic factors delivered by genetransfer will aid in the acceleration and augmentationof fracture healing. Use of bone morphogeneticproteins in combination with osteogenic cells willprovide clinicians with new tools for the treatment offractures. One can envisage a scenario where bonemarrow is harvested from the patient, geneticallymodified by insertion of therapeutic genes and thenre-implanted in the fracture site (Figure 1).

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Christopher Niyibizi† & Myung Kim†Author for correspondenceUniversity of Pittsburgh School of Medicine, MusculoskeletalResearch Center, Department of Orthopaedic Surgery, 200 LothropStreet, RM C313, Pittsburgh, PA, 15213, USATel.: +1 412 648 1091; Fax: +1 412 648 8412;E-mail: [email protected]



novel approaches to fracture healing

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