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Biomaterials 27 (2006) 1851–1858

www.elsevier.com/locate/biomaterials

Review

Bioluminescent imaging: Emerging technology for non-invasive imagingof bone tissue engineering

Jan de Boera,�, Clemens van Blitterswijka, Clemens Lowikb

aInstitute for Biomedical Technology, University of Twente, Prof. Bronkhorstlaan 10D, 3723 MB, Bilthoven, The NetherlandsbDepartment of Endocrinology, Leiden University Medical Centre, Leiden, The Netherlands

Received 27 April 2005; accepted 26 September 2005

Available online 20 October 2005

Abstract

Bone tissue engineering is a multidisciplinary research area in which new strategies are developed to treat patients with large bone

defects as occurring during e.g. hip revisions, upon trauma or in spinal fusions. In vivo evaluation of bone formation in animal models is

highly relevant for graft evaluation but is time-consuming, invasive and difficult to quantify. As a consequence, most in vivo studies

ignore the dynamic nature of bone regeneration and the molecular processes underlying it. In vivo bioluminescent imaging (BLI) is a

relatively young research field with great potential that may overcome these problems. BLI encompasses non-invasive imaging of

luciferase gene activity using cooled charge coupled device cameras in luciferase transgenic animals or in grafted, luciferase transgenic

cells. The imaging procedure is technically simple and quantifiable. Because luciferase expression can be put under the control of tissue-

specific regulatory elements, BLI allows non-invasive imaging of processes highly relevant to bone tissue engineering like differentiation,

apoptosis, vasculogenesis and inflammation. In this review, we describe the basic principle of BLI and discuss transgenic animals and

constructs currently available for application in bone tissue engineering. Furthermore, we reflect on technical developments that will

make BLI even more promising for future application in bone tissue engineering research.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Bone tissue engineering; Molecular imaging

Contents

1. Bone tissue engineering: a technological meltingpot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1851

2. Clogging the pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1852

3. Bioluminescent imaging (BLI) in rodents: an emerging technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1852

4. Imaging of biological processes relevant to bone regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1854

4.1. Cell numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1854

4.2. Osteogenic differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1854

4.3. Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1855

4.4. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1856

4.5. Vasculogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1856

4.6. Signal transduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1856

5. Current limitations and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1857

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1857

e front matter r 2005 Elsevier Ltd. All rights reserved.

omaterials.2005.09.034

ing author. Tel.: +3130 2295154; fax: +31 30 2280225.

ess: j.deboer@tnw.utwente.nl (J. de Boer).

1. Bone tissue engineering: a technological meltingpot

Bone tissue engineering is a relatively young, rapidlyevolving and innovative research field, which combines a

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multitude of scientific disciplines such as materials science,stem cell biology and molecular biology in order toengineer improved grafts for the treatment of large bonedefects. Cross-disciplinarity is the rule rather than theexception. Bone tissue engineers are increasingly aware thatmaterials traditionally used for bone regeneration, such asceramics, titanium and polymers are not merely there todeliver and support grafted cells or provide mechanicalstrength. They can play a direct role in modulating tissueformation by interacting with endogenous or implantedcells, as vehicle for controlled release of bioactivecompounds and by creating the proper microenvironmentfor tissue growth [1–5]. Recent years have seen a surge inknowledge of the molecular pathways controlling bonegrowth and regeneration. Every step in the fascinatingcellular cascade leading from a multipotent mesenchymalstem cell to a mineralising osteoblast is controlled bydifferent molecular cues, i.e. growth factors, extracellularmatrix proteins, cytokines and hormones. Translation ofmolecular knowledge into improved bone grafts is anexciting new challenge in bone tissue engineering research[6]. A classical ‘‘from-bench-to-bed’’ example is theidentification and clinical application of bone morphoge-netic proteins [7,8]. Other examples of applied molecularknowledge include incorporation into or coating ofbiomaterials with bioactive groups such as chitosan [9]and RGD peptides [10] for enhanced cell binding, andcontrolled release of bone-inducing growth factors frommaterials [11–13]. Besides their direct application in boneregeneration technology, genes involved in osteogenesis arealso useful molecular markers for analysis of the variousstages of bone formation.

2. Clogging the pipeline

Newly designed bone grafts have to be tested for theirbiological performance. To assess applicability, each newgraft should be tested in a system as close as possible to theclinical reality, but for practical reasons the test systemshould allow rapid screening of many variables at low costand in a short period of time. Evidently, these viewsconflict and in practice, product development starts with invitro biological assays, to proceed with in vivo studies inrodent models, then larger animal models and finallyclinical trials in human beings. The great advantage of invitro biological assays is that they allow a degree ofautomation and are therefore suitable for medium to highthroughput biomaterials research [14], gene discovery[15,16] and cellular assays [17]. However, in vitro assaysare mostly limited to cellular research because it is difficultto mimic the complexity of tissue formation, which is a 3-dimensional (3D) interplay between different cell types, in atest tube. In vivo bone formation by stem cells is mostlystudied by seeding the cells onto biomaterials andsubsequent ectopic implantation in the back of immune-deficient mice. Ectopic implantation is a simple surgicalhandling and immune-deficient mice allow implantation of

xenogenic cells and materials. In this milieu, mesenchymalstem cells produce bone matrix, blood vessels will infiltratethe tissue and bone marrow formation is supported, whichallows investigation of a number of highly relevant aspectsof bone tissue engineering. A clinically even more relevantimplantation site is bone itself but orthotopic implanta-tions are technically more difficult to perform and moreexpensive because one has to rely on larger animal modelssuch as rats, goats or rabbits. Although biologically morerelevant, orthotopic and ectopic bone formation modelslack the versatility of in vitro assays due to the limitedbiological readout and time-consuming analysis. Whereasin vitro osteogenic differentiation can be easily followedthrough time by quantitative PCR, flow cytometry orELISA, in vivo analysis of bone formation mostly dependson histological examination at single time points andtedious in situ hybridisation or immunohistochemistry.Evaluation at different time intervals surmounts toexcessive animal usage and time-consuming histologicalanalysis. Due to these limitations, many in vivo studiesignore the dynamic molecular and cellular processesleading to repair of bone defects. Bioluminescent imagingprovides a technological platform that allows non-invasiveimaging of marker gene expression. Thus, all aspects ofbone formation can be followed in individual animalsfrom the time of implantation until the moment whennewly formed bone is remodelled and fully integrated (seeTable 1).

3. Bioluminescent imaging (BLI) in rodents: an emerging

technology

The recent surge in BLI research is spurred by thedemand of cancer biologists to image tumour growth andmaturation and the efficacy of therapy in vivo (for reviewson BLI and molecular imaging see [18–20]. Biolumines-cence refers to the enzymatic generation of visible light byliving organisms. The most commonly used bioluminescentreporter gene for research purposes is luciferase from theNorth American firefly Photinus pyralis. The enzymecatalyses the transformation of its substrate D-luciferininto oxoluciferin, in an oxygen and ATP-dependentprocess, leading to emission of photons (see Fig. 1).Biological sources of light have sufficient intensity to crossanimal tissues, including bone, provided that the light has awavelength of more than 550 nm. The firefly luciferase/luciferin reaction emits photons at wavelengths from 500 to620 nm and is therefore suitable for external detection.Apart from fulfilling this criterion, another importantfeature is that the substrate luciferin diffuses within fewminutes from intravenous or peritoneal injection through-out the entire body of small animals and is rapidly taken upby cells [21]. The quantitative relationship between geneexpression and the detected signal is influenced by severalfactors. First of all, the luciferin substrate must be availablewithin the cells, which is influenced by factors such astechnical skills of the experimentor to inject the substrate

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Table 1

Transgenic reporters currently available for BLI of bone tissue engineering

Name Regulatory element Application Reference

Transgenic mice

hOC-luc Human osteocalcin promoter Matrix deposition [31]

colI(a1)-luc Bone-specific enhancer of the mouse collagen (a1) I gene Matrix deposition [33]

BSP-luc Mouse BSP promoter Matrix deposition [36]

VEGFR2-luc Mouse VEGFR2 gene promoter Angiogenesis [46]

iNOS-luc Mouse iNOS gene promoter Inflammation [40]

NFkB-luc 3 NFkB site from the Igk light chain promoter Inflammation [39]

ER-DEVD-luc Caspase cleavage sequence Apoptosis [41]

Transgenic constructs

Retro-luc Cytomegalovirus promoter Cell tracking [28]

BRE-luc BMP responsive element of the ID1 gene Osteo-induction, BMP CDD [30]

WRE-luc TCF/LEF binding sites Wnt signalling [54]

Hes1-luc Hes1 gene promoter Differentiation [52]

Fech-luc Ferrochelatase promoter Hypoxia [48]

HRE-luc 5 hypoxia responsive elements from the VEGF gene Hypoxia [49]

Fig. 1. Biological principle of bioluminescent imaging of luciferase expression. (A) Firefly luciferase converts the substrate luciferin into oxyluciferin and

light. Tissue-specific transcription factors bind to regulatory sequences cloned directly adjacent to the luciferase gene, resulting in tissue specific expression

of luciferase, which can be visualised by injection of luciferin and detection of the emitted light by CCCD cameras. (B) Image of a transgenic mouse during

pro-estrous expressing the luciferase gene under the control of an estrogen responsive element. Note the intense staining of the ovaries, which subsides

during estrous.

J. de Boer et al. / Biomaterials 27 (2006) 1851–1858 1853

but also by the degree of vascularisation of the targettissue. For example, we recently used BLI as a tool tomeasure enhanced luciferin perfusion in a vascularisedtissue construct [22]. Furthermore, light scattering occursas it travels through the body. As such, it is difficult tocompare the relative light intensities emitted from differentorgans because light scattering depends on the character-

istics of the tissue. Recent technical advances for imagingweak visible light sources using cooled charged coupleddevice (CCCD) cameras, peltier cooled detectors andmicro-plate channel intensifiers allow detection and quan-tification of bioluminescent emission from inside the tissuesof the animals. This technical development made it possibleto monitor gene expression in living animals via a luciferase

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Fig. 2. BLI of transgenic cancer cells. 1� 105 luciferase transgenic MD-

231 cells were injected in the left hart ventricle and bone metastases were

imaged using BLI. Photon intensity was indicated using pseudo-colouring.

Note the metastasis throughout the body of the mouse.

J. de Boer et al. / Biomaterials 27 (2006) 1851–18581854

reporter gene linked to specific promoters [21,23] (Fig. 1).For instance, we generated mice transgenic for theluciferase gene under the control of an estrogen-responsiveelement (ERE-luc) [24]. Luciferase activity in individualmice could be followed throughout the estrous cycle and apeak of luciferase activity was observed during proesterusin reproductive tissues (Fig. 1). Using ERE-luc mice, wewere able to monitor estrogen receptor activity dynamics ina tissue-specific and quantitative way, demonstrating thepower of BLI. Besides imaging of transgene expression intransgenic animals, BLI can also be used to follow in realtime the fate of luciferase transfected cells injected in livinganimals, an application which is widely used in cancerresearch [25]. One of the many advantages of BLI is that itis non-invasive, and therefore allows investigations in thesame animal at different time points. Accordingly, thenumber of animal groups in the experimental setting isgreatly reduced and the data variation is reduced becausethe signal is sequentially detected in the same animal.

Detection of the bioluminescent activity in vivo of theluciferase enzyme as a transcriptional reporter facilitatesrapid and repetitive screening for both the presence andfunction of transgenes in intact living mice [26]. Below, wewill discuss recent developments in the field of BLI thathave brought the technique within the reach of bone tissueengineering research.

4. Imaging of biological processes relevant to bone

regeneration

4.1. Cell numbers

Bone tissue engineering often includes seeding ofosteogenic cells onto biomaterials and co-implantation inan ectopic or orthotopic site. Survival of the cells relies onboth cell and biomaterial properties and it would be veryvaluable to assess survival and proliferation of implantedcells for a prolonged period of time in the same animal. Forthis purpose, transgenic cells can be prepared expressingthe luciferase gene under the control of a constitutivelyactive promoter. Using viral transduction, virtually everycell type used in bone tissue engineering, including MSCs,can be stably transduced with a luciferase gene and thusfollowed in real time upon implantation. Luciferasetransgenic cells have been extensively used in cancerresearch, for instance to monitor the efficacy of drugtreatment on the survival of implanted cancer cells or toanalyse tumour metastasis. For instance, we implantedluciferase transgenic mammary carcinoma cells into the leftcardiac ventricle of mice and during a 36 days follow-upstudy we were able to trace bone marrow progression andmetastasis of �0.5mm3 (see Fig. 2), which is below thelimit at which tumours need angiogenesis for furthergrowth and well before radiological osteolysis can beobserved [27]. One report demonstrates the proof ofconcept for the use of luminescent cells in the field oftissue engineering. Blum et al. [28] used a stably transfected

fibroblast cell line expressing a luciferase reporter constructunder the control of a constitutively active viral promoter.The transgenic cells were seeded on a titanium mesh usedfor bone tissue engineering purposes and implantedsubcutaneously in nude mice. Using BLI, Blum et al. wereable to monitor cell survival over a period of 28 days.

4.2. Osteogenic differentiation

The tissue- and time-specific expression of genes thatdelineate the process of osteogenic differentiation can bemonitored by driving the expression of luciferase byspecific regulatory elements. For instance, Korchynski etal. [29] identified a DNA sequence in the promoter of theID1 gene, designated the bone morphogenetic proteinresponsive element (BRE), which responds uniquely toBMP stimulation. Monteiro et al. [30] used the BRE todrive lacZ in transgenic mice and luciferase in embryonicstem cells. They were able to detect lacZ expression inmany tissues in which BMP is known to be active and theyshowed a BMP dose–responsive upregulation of luciferaseactivity in ES cells. Because activation of BMP signallingmarks the onset of osteogenic differentiation, BRE-luciferase mice and cells can in the future be used to assaythe earliest events of osteogenic differentiation.A later phase of differentiation, i.e. osteoblast matrix

production, can be monitored by luciferase under thecontrol of the osteocalcin promoter. Using mice transgenicfor the human osteocalcin promoter driving the luciferasegene [31], Iris et al. [32] observed luciferase expression inliving mice upon bone formation induced by fracturehealing or bone marrow ablation. Interestingly, luciferaseexpression was highly induced upon injection of vitaminD3, a compound known to directly activate the osteocalcingene (Fig. 3). Similarly, mice transgenic for the bone-specific regulatory element of the collagen type I gene (Col(a1) I-luc mice) were treated with parathyroid hormone orprostaglandin E2, which are known bone anabolic agents[33]. In both cases, enhanced luciferase expression wasobserved in tissue extracts demonstrating that Col (a1)

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Fig. 3. BLI of osteocalcin expression. BLI of a hOC-luc mouse under basal conditions and after bone marrow ablation, which is known to result in very

strong and local bone formation during the first 10–12 days after ablation. In the last frame, a mouse is shown after injection of vitamin D3, a known

stimulator of osteocalcin expression.

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I-luc mice are useful to study bone anabolic compounds invivo. A third gene involved in matrix maturation is bonesialoprotein. Based on its expression profile and biochem-ical properties, BSP is thought to be involved in the initialphase of mineral formation. A series of papers describingthe generation and characterisation of mice expressing theluciferase gene under the control of the bone sialoproteingene promoter have been published [34–36]. Luciferasegene expression faithfully follows the expression of theendogenous gene and transgene expression could bemodified by bone anabolic agents. For instance, BSP-lucmice treated with the glucocorticoid dexamethasonedisplayed enhanced luciferase expression [35]. Conversely,mice fed on a vitamin D3-deficient diet exhibited enhancedluciferase expression because the mice suffer from a blockin osteogenesis at the osteoblast stage [34]. As a conse-quence, the number of osteoblasts accumulates andluciferase activity is enhanced. Further useful models tomonitor osteogenesis can be expected as the result of therecent identification of genes, which are uniquely expressedduring the osteocyte stage [37]. Osteocyte-specific regula-tory elements in those genes can be employed in non-invasive imaging of osteocyte formation.

The above-mentioned transgenic mice will be extremelyuseful in osteo-induction studies, e.g. to quantify ectopicbone formation by recombinant BMP in controlleddelivery studies or in bone formation induced by deminer-alised bone matrix. MSCs isolated from these mice can be

used for real-time imaging of osteogenic differentiation inbone tissue engineering.

4.3. Inflammation

An important aspect in the application of biomaterials isa putative immune response evoked by implantation of thegraft. Moreover, implantation of biomaterials increases thechance of bacterial infection both during surgery also post-implantation when biomaterial surfaces may offer asubstrate for bacteria to attach to and proliferate. There-fore, real-time monitoring of immunological parameters atthe site of implantation, which is currently assessed byhistological examination of explanted materials, is highlyrelevant.BLI can be applied in this area in a number of ways. BLI

is used to monitor biofilm formation using luciferasetransgenic bacteria [38], although no reports are known sofar in which this technique is applied to biomaterialsresearch.Moreover, BLI can be used to monitor inflammation by

driving luciferase with inflammation-specific regulatorysequences. For instance, Carlsen et al. generated miceexpressing luciferase under the control of the regulatorysequences of the NFkB gene, which in its turn is under thedirect control of TNFa, a key cytokine produced duringinflammation. Using NFkB-luciferase mice, Carlsen et al.[39] monitored osteoarthritic inflammation induced by

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injection of bacterial lipopolysacharide, and quantified thetherapeutic potential of dexamethasone treatment of thearthritic lesion. Using a similar approach, iNOS-luciferasemice were used to image zymosan-induced arthritis in theknee joint [40]. Like NFkB, iNOS is induced duringinflammation and therefore both mouse models might beused for biocompatibility studies of newly designedmaterials.

4.4. Apoptosis

An unwanted side effect of implanted materials iscytotoxicity to the cells surrounding the implant. Apopto-sis, i.e. programmed cell death, can be used as a marker forcytotoxicity.

Laxman et al. developed an elegant tool to monitorapoptosis based on the activity of caspases, which aresequence-specific proteases specifically activated uponinitiation of apoptosis. They generated a recombinant geneencoding a chimeric protein of luciferase fused to a domainof the estrogen receptor (ER). The ER domain efficientlysilences luciferase reporter activity [41] but the inactivechimeric protein can be activated upon proteolytic cleavageof a caspase recognition site separating luciferase and thesilencing domain. Laxman et al. [41] describe how theywere able to monitor apoptosis in tumourigenic cellsimplanted in nude mice and similar studies could beperformed with implanted materials. The paper furtherunderscores the versatility of this approach. Using thesame methodology, one could monitor the activity of anysequence-specific protease such as e.g. metalloproteasesinvolved in arthritis, simply by changing the caspaserecognition sequence with other protease sensitive se-quences. Another apoptosis model makes use of the factthat the p53 protein accumulates upon apoptosis andactivates gene expression through p53-responsive elements.The specificity of these regulatory sequences has beendemonstrated in vivo by PET imaging of DNA damage-induced apoptosis [42] and in a bioluminescent xenograftmodel of human colon carcinoma cells [43].

4.5. Vasculogenesis

Bone is a well-vascularised tissue and vasculogenesisduring bone regeneration serves two purposes. First, itprovides nutrition to the implanted cells and adjacenttissue and, secondly, it presents the molecular cues requiredfor the final step of endochondral bone formation: invasionby MSCs of the fracture area and replacement ofhypertrophic cartilage tissue by bone. A number oftechniques exist that monitor vasculogenesis indirectly byimaging of perfusion of the implant by an injected tracer,i.e. using MRI [44] or CT [45]. Direct BLI of vasculogenesisis feasible using endothelial specific promoters drivingluciferase expression. For instance, Zhang et al. [46]describe vascular endothelial growth factor receptor 2(VEGFR2)-luciferase mice. In a cutaneous wound healing

model, VEGFR2-luc expression was monitored in thewound tissue. Dexamethasone suppressed luciferase activ-ity, concomitant with delayed healing and impairedangiogenesis. Alternatively, endothelial specificity can beprovided by the Tie2 promoter. Using Tie2-driven GFPexpression in whole mounts of mouse retina, Otani et al.[47] could clearly delineate the eye vasculature. In thefuture, Tie2-luciferase and VEGR2-luciferase mice mightbe employed to monitor blood vessel ingrowth in bonegrafts.In close relation to vasculogenis, the nutritional status of

implanted cells might be assessed by BLI of hypoxia, amajor problem in tissue engineering of large bone grafts.Both the ferrochelatase and VEGF gene promoter respondto hypoxia-inducible factor and are clearly regulated byhypoxic conditions [48,49]. In one report, a hypoxia-responsive element was used to drive expression ofluciferase and enhanced luciferase activity was seen inischemic tissue [50].

4.6. Signal transduction

As discussed before, bone tissue engineering increasinglyincorporates molecular cues into the graft, i.e. growthfactors, small molecules and adhesion proteins to promotetissue regeneration. BLI can be used to monitor thebiological activity of these molecules in vivo because anumber of DNA sequences are known which activate genetranscription as a direct consequence of the compound’sbiological activity. Examples include the BMP-responsiveelement identified in the ID-1 gene; the TCF/LEF-bindingsites responding to Wnt signaling [51]; the promoter of theHes1 gene, which is a direct target gene of Notch signalling[52]; and the responsive elements to either the estrogen,glucocorticoid or retinoic acid receptors [19 and referencestherein]. For example, the previously discussed BRE-luciferase construct can be employed to trace the biologicalactivity of BMP in demineralised bone matrix or recombi-nant BMP. A number of reports describe incorporation ofBMP into materials for local release but, so far, the in vivorelease profile and biological activity are black boxes andefficacy can only be scored in post-mortem evaluation ofbone formation. Using BRE-luciferase transgenic mice orcells one could on-line monitor the biological efficacy ofreleased BMPs. Similarly, the Wnt signalling cascade playsan important role in bone biology and strategies aimedat enhancing Wnt signalling during bone regenerationare anticipated. We recently discovered that humanmesenchymal stem cell proliferation is enhanced by theWnt-activating drug lithium [53] and, to this end, weincorporated lithium into calcium phosphate coatings(unpublished data). Wnt activates target genes throughthe well-characterised TCF/LEF-binding sites (WRE) andnumerous papers describe the use of WRE-luciferaseconstructs to monitor Wnt activity in vitro [54,55]. Tomonitor the biological activity of our coatings in vivo, weare currently generating WRE-luciferase transgenic mice.

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5. Current limitations and future directions

The current BLI systems use photographic principles tocapture light emitted from the animals at sites whereluciferase is expressed. Photons are detected at the surfaceof the animal in 2D, also known as planar imaging, thesimplest technique for detecting optical reporter moleculesin vivo. Photon attenuation, however, is strongly nonlinearas a function of depth and of the optical heterogeneity oftissue, which hampers signal quantification. Planar imagingis further complicated by the inability to resolve depth andby tissue scattering and absorption, which limits spatialresolution. A way to circumvent the problems of 2D planarBLI would be a combination of multi-view angle imaging(bioluminescence tomography) with a priori informationon tissue heterogeneity. This can now be achieved using thenewly developed 3D BLI systems from Xenogen andBerthold Technologies. If these 3D images are combinedwith 3D spatial (tissue) information obtained by CT orMRI, algorithms can be developed to more preciselycorrect for photon scattering and absorption due to tissueheterogeneity and depth.

Besides advances in the optimisation in imaging equip-ment and software, recent years have also seen a steadyincrease in the number of transgenic reporter constructs,which express luciferase under the control of specificregulatory elements and the generation of transgenic cellsand animals from them. The emergence of BLI in rodentslikely spurs the generation of many more and they will beapplicable in three ways. First, reporter constructs can beintroduced into any cell type used in bone tissue engineer-ing by means of e.g. viral transduction, which allowsus to acquire molecular data from clinically relevantcell types in vivo, i.e. human mesenchymal stem cells.Secondly, cells can be isolated from transgenic mice.Efficient protocols exist to isolate mesenchymal stemcells from bone marrow and fat tissue, and embryonicstem cells from pre-implantation blastocysts. Finally,parameters relevant to the bone regeneration processcan be monitored in transgenic animals. Because oftheir small size, it is very difficult to use mice for orthotopicimplantation but rats are frequently used for this purpose.The current advances in rat genetics, such as the comple-tion of the rat genome project [56] and strongly improvedtechnology to generate transgenic rats [57], bring BLIof orthotopic bone regeneration within reach. Otheradvances in the field that are likely to further reducethe threshold to apply BLI in bone tissue engineeringare improved imaging (software, cameras) with moresensitive and 3D imaging modes. Finally, luciferasefrom other species becomes available which emit lightat different wavelengths. Simultaneous imaging of differentparameters thus becomes possible [58,59]. In conclusion,we believe that BLI is knocking on the door of bonetissue engineering and can strongly contribute to ourunderstanding of the in vivo dynamics of bone tissueengineering.

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