Cell-based resorption assays for bone graft substitutes

Acta Biomaterialia 8 (2012) 13–19

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Review

Cell-based resorption assays for bone graft substitutes Ziyang Zhang, José T. Egaña, Ann K. Reckhenrich, Thilo Ludwig Schenck, Jörn A. Lohmeyer, Jan Thorsten Schantz, Hans-Günther Machens, Arndt F. Schilling ⇑ Klinik und Poliklinik für Plastische Chirurgie und Handchirurgie, Klinikum Rechts der Isar, Technische Universität München, München, Germany

a r t i c l e

i n f o

Article history: Received 9 May 2011 Received in revised form 16 September 2011 Accepted 20 September 2011 Available online 24 September 2011 Keywords: Biodegradation Surface analysis Osteoclasts Biomimetic material In vitro test

a b s t r a c t The clinical utilization of resorbable bone substitutes has been growing rapidly during the last decade, creating a rising demand for new resorbable biomaterials. An ideal resorbable bone substitute should not only function as a load-bearing material but also integrate into the local bone remodeling process. This means that these bone substitutes need to undergo controlled resorption and then be replaced by newly formed bone structures. Thus the assessment of resorbability is an important first step in predicting the in vivo clinical function of bone substitute biomaterials. Compared with in vivo assays, cell-based assays are relatively easy, reproducible, inexpensive and do not involve the suffering of animals. Moreover, the discovery of RANKL and M-CSF for osteoclastic differentiation has made the differentiation and cultivation of human osteoclasts possible and, as a result, human cell-based bone substitute resorption assays have been developed. In addition, the evolution of microscopy technology allows advanced analyses of the resorption pits on biomaterials. The aim of the current review is to give a concise update on in vitro cell-based resorption assays for analyzing bone substitute resorption. For this purpose models using different cells from different species are compared. Several popular two-dimensional and threedimensional optical methods used for resorption assays are described. The limitations and advantages of the current ISO degradation assay in comparison with cell-based assays are discussed. Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Bone is a rigid organ which is constantly remodeled. Old or damaged bone is repaired by replacing it with newly formed bone structures. Several kinds of cells are involved in this remodeling process, including osteoblasts, osteocytes, lining cells and osteoclasts [1,2]. In reconstructive bone surgery bone replacement is of great importance when critical skeletal defects occur [3]. Bone grafting (autograft or allograft) is still the gold standard due to the good osteoinductive and osteoconductive performance and excellent biomechanical properties [4]. However, the shortage of skeletal donor tissue, the growing number of bone grafting procedures worldwide and also graft resorption have stimulated the development of bone graft substitutes [5–7]. The desired outcome of a bone grafting procedure is a return to function for the specific patient. This can be achieved in some of the cases with non-resorbable materials, typically made of metals or ceramics [8]. These implants, however, will not grow with growing children and are in the long run subject to material fatigue, and loosening at bone sites with high cyclic loading [9]. Therefore, non-resorbable materials are used successfully in older patients and cranial surgery, ⇑ Corresponding author. Tel.: +49 89 4140 2171; fax: +49 89 4140 7336. E-mail address: [email protected] (A.F. Schilling).

however, in young and active patients resorbable implants would have the advantage of being able to adapt to changing environmental needs by integrating into the bone remodeling process. The ideal resorbable bone graft substitute should have similar biomechanical properties to the autologous bone at the time of implantation, and should then be remodeled into new bone. This means bioresorption of the material and the formation of new bone should be balanced to secure functional bone mechanics (‘‘smooth assimilation’’) [10–12]. There are a variety of materials used for resorbable bone substitutes, including calcium carbonate, calcium sulfate, calcium phosphates, magnesium, iron, titanium and polymers (synthetic and natural) [13,14]. However, some of the so called ‘‘resorbable’’ bone graft substitutes can still be detected years after in vivo transplantation [15–17]. On the other hand, increased resorption without sufficient bone formation can lead to deleterious results [18,19]. Therefore, evaluation of resorbability of bone substitutes is an important step before in vivo clinical application. So far, bone grafting substitutes are usually evaluated in vivo in different animal models, such as dogs [20], mice [21], rats [22], rabbits [23], pigs [24], sheep [25], goats [26], horses [27] and monkeys [28]. Cell-based, especially human cellbased, resorption assays can be used to lower the number of these animal experiments and thus should be part of the preclinical analysis procedure.

1742-7061/$ - see front matter Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.09.020

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2. ISO medical biomaterials resorption assay In vitro the degradation of medical biomaterials, including bone graft substitutes, is usually evaluated according to DIN EN ISO 10993 [29]. For ceramic biomaterials DIN EN ISO 10993-14 applies. It consists of two parts: (1) degradation in an extreme solution, typically citric acid buffer solution (pH 3), which represents the worst possible environment in vivo for the material; (2) degradation in a solution that represents the common in vivo environment, typically Tris–HCl buffer with a pH of 7.4 ± 0.1. The main advantage of such an approach is a standardized testing procedure that can be easily applied worldwide. This allows comparison between the results of different groups from different countries. However, some so called ‘‘fully resorbable’’ bone graft substitutes which were tested under ISO standard conditions were not completely resorbed in vivo even a long time after implantation [15,16,30]. This may be due to the fact that in vivo biodegradation involves not only body fluids but also other components like body movements, enzymes, proteins and cells. Consequently, cell-based assays were developed to improve preclinical bone graft evaluation. 3. Cell-based assays In vivo hard tissues such as bone and tooth are mainly degraded by a specialized cell type, the osteoclast [31]. There are reports that macrophages and monocytes, which share a common origin with osteoclasts in the hematopoietic lineage, may also be involved in the bone substitute resorption process [32–34]. In vitro there are two kinds of systems for cell-based resorption assays: three-dimensional (3-D) tissue culture and two-dimensional (2-D) cell culture. 3-D tissue culture is used for the analysis of in situ resorption of bone structures and the measurement of calcium or hydroxyproline release [35,36]. Although the local environment is preserved in such assays, the influence of the systematic circulation is removed. Moreover, it is impossible to evaluate different bone substitute biomaterials other than fetal or neonate tissues. This makes 2-D cell culture more popular for evaluating different bone grafting substitutes, especially for high throughput analysis. The physiological localization of osteoclasts in the bone and their ability to resorb different biomaterials [12,37] make the osteoclast the primary cell type for such cell-based resorption assays. 3.1. Cell sources for osteoclastic resorption assays 3.1.1. Primary osteoclasts Several early studies have used different kinds of animal models for the isolation of primary osteoclasts (Table 1). Most of those

studies are based on the fact that osteoclasts from the bones of neonatal animals are relatively abundant and therefore easy to isolate [38]. In 1984 Chambers and colleagues evaluated bone resorption with cortical bone slices and rabbit osteoclasts. Osteoclasts were disaggregated from neonatal rabbit long bones by fragmenting the bone structure in HEPES-buffered medium. The cells were then seeded on the cortical bone slices [39]. Similar isolation procedures were then transferred to a neonatal rat and a neonatal chicken [40]. Interestingly, Jones et al. found that osteoclasts isolated by this approach showed no differences in species (rat, chicken and rabbit) with regard to substance resorption [41]. Apart from the neonatal animals, bone from laying hens that had been fed a low calcium diet for 1 [42] or 4 weeks [43] was used as another source. This approach was considered by some researchers to be the richest source for the isolation of large numbers of primary osteoclasts [43,44]. 3.1.2. Tumor osteoclasts In humans primary osteoclast-like cells are usually isolated from giant cell tumor tissue [45]. This kind of tumor is relatively rare and is usually not malignant [46]. Although the tumor often occurs in bone tissue it can also be found in other tissues, such as parotid [47], pancreas [48] and urinary bladder [49]. Compared with the isolation of human primary osteoclasts from other sources, osteoclast-like cells obtained by this approach are usually abundant [45,50]. However, due to the rarity of the disease it is not easy to obtain sufficient cells for regular experiments. Furthermore, due to the tumor origin of these cells their behavior seems particularly aggressive and some cell features are different from primary human osteoclasts [51,52]. For example, the estrogen receptor is missing from osteoclasts from human giant cell tumor of bone [51] and a significant decrease in TGF-b expression in multinucleated cells from giant cell tumor patients could be detected compared with corresponding cells from normal bone [53]. 3.1.3. Osteoclast/osteoblast co-culture Both osteoclasts and osteoblasts are involved in the bone remodeling process, and their functions are closely linked. Thus co-culture of osteoclasts and osteoblasts has also been used for bone substitute resorption assays. Compared with osteoclasts, osteoblasts are relatively easy to isolate and suitable for long-term in vitro culture. After the first successful isolation of osteoblasts, in 1974 [54], osteoblasts were successfully isolated from several different species, including human [55–57]. Growth factors secreted by osteoblasts are important in modulating osteoclast differentiation in vivo and therefore osteoclast resorption ability [58]. Teti et al. used a serum-free co-culture system with osteoclasts and

Table 1 Typical sources of osteoclasts/osteoclast-like cells. Species

Age

Cell source

Osteoclast type

Differentiation methods

References

Chicken

Long bone from low calcium diet chicken Calvaria (skull) Bone marrow cells

Primary osteoclasts

No differentiation

Oursler et al. [43]

Mouse Mouse

Chicken hatchlings Neonatal Unknown

Primary osteoclasts Differentiated osteoclasts

Yasuda et al. [64] Lacey et al. [99]

Mouse

6–15 weeks

Spleen cells

Differentiated osteoclasts

Mouse Rabbit

Unknown Neonatal

RAW264.7 Minced bone cells from long bones

Rat

Neonatal

Long bones

Osteoclast-like cells Primary cells + differentiated cells Primary osteoclasts

No differentiation (1) Coculture with osteoblast-like cells2) RANKL + MCSF (1) Coculture with osteoblast-like cells2) RANKL + MCSF RANKL Vitamin D3 and 1,25(OH)2D3 No differentiation

Human

Unknown

Human

Unknown

Human

Unknown

Human peripheral blood mononuclear cells Human peripheral blood mononuclear cells Bone marrow mononuclear cell

Yasuda et al. [64] Cuetara et al. [68] David et al. [101]

Differentiated osteoclasts

RANKL + M-CSF

Hoebertz and Arnett [38] Schilling et al. [31]

Differentiated osteoclasts

Coculture with bone-derived osteoblasts

Atkins et al. [100]

Differentiated osteoclasts

Coculture with bone-derived osteoblasts

Atkins et al. [100]

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osteoblasts separated by a 0.45 lm porous membrane permeable to soluble molecules to evaluate the effects of osteoblasts on osteoclast resorption ability. Unlike control cells (periosteal cells and skin fibroblasts), osteoblasts or chick calvaria which contain osteoblasts could significantly enhance the resorption ability of osteoclasts [59]. tumor necrosis factor-related activation-induced cytokine (TRANCE), now known as receptor activator of nuclear factor B ligand (RANKL) or tumor necrosis factor ligand superfamily member 11 (TNFSF11), was found to be strongly related to osteoblast-mediated activation of osteoclastic bone resorption [60]. Characterization of the RANKL/osteoprotegerin (OPG)/macrophage colony-stimulating factor (M-CSF) signaling pathway in osteoclast differentiation [61] and recombinant production of these factors now allows direct differentiation of osteoclasts from hematopoietic precursor cells. 3.1.4. Differentiation of mononuclear precursors using RANKL and MCSF Both RANKL and M-CSF are secreted by osteoblasts in vivo, activating osteoclast differentiation [62]. M-CSF is a hematopoietic growth factor which is involved in the proliferation and differentiation of monocytes. Although indispensable for osteoclast differentiation, high concentrations of M-CSF actually inhibit osteoclast formation in vitro [63]. The identification of RANKL as a differentiation factor for osteoclasts [64] and its recombinant production has had a major impact on cell-based resorption assays. Isolation of mononuclear precursors from different sources and species and differentiation into osteoclasts has since become more and more popular in cell-based resorption assays. 3.1.5. Raw 267.4 RAW 267.4 is a mouse leukemic monocyte macrophage cell line. It was established in mice from the ascites of a tumor induced by intraperitoneal injection of Abselon leukemia virus (A-MuLV) [65]. RANKL is capable of activating nuclear factor jB (NF-jB) and inducing osteoclastic differentiation in RAW 264.7 cells [66,67]. In addition, the generation of osteoclast-like cells from RAW 264.7 cells does not require any co-administration of MCSF, which is a major advantage of RAW 264.7 over other cells [68,69]. Although RAW 264.7 cells are well characterized regarding macrophage behavior, there are still concerns about using such cells as precursors for osteoclast-like cells due to the heterogeneity of macrophages [69,70]. Nevertheless, RAW 264.7 cells can be an easy source for the generation of osteoclast-like cells for preliminary resorption assays. 3.2. Methods for the evaluation of resorption Cell-based bone resorption assays are mostly conducted by culturing osteoclasts on flat slices of different bone substitutes. After a

certain period of time the osteoclast resorption lacunae or pits are then analyzed (pit assay). Both 2-D and 3-D measurements are used for these quantifications. Some classical and advanced methods for the evaluation of resorption are listed in Table 2 in chronological order, and are also described in the text below. The pioneer studies for cell-based assays were conducted in the 1980s. Two basic measurement techniques, which are still used today, were introduced: light microscopy (LM) after toluidine blue staining of the resorbed specimen and resorption area measurements by scanning electron microscopy (SEM) [39,71]. After measuring the pit area and number this can be divided by the number of osteoclasts to obtain an index for single cell activity on the respective material [72]. By normalizing the results to a co-cultured standard material (dentin) a relative resorption coefficient (RRC) can be derived, which allows comparisons between different materials [31].

3.2.1. Light microscopy (LM) For 2-D measurements LM is still the most commonly used method for analyzing cellular resorption. The probable reasons for this are the relatively low costs of the equipment and the consequent wide availability, together with the relative ease of the procedure. LM can be used to first identify the number and size of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (Fig. 1) and evaluate the number of nuclei per cell. Then, after removal of the cells, resorption pits in the same area can be identified with a simple toluidine blue stain [73] (Fig. 2) and quantified using image analysis software. However, the LM resorption assay is critically dependent on the possibility of staining the lacunae. This is easily done with materials like bone or dentin, where the resorption process uncovers collagen fibrils embedded in the calcified matrix. These free collagen ends stain much better than the surrounding hydroxyapatite and therefore give good contrast between resorbed and non-resorbed areas. Artificial bone substitute biomaterials do not typically contain such a structure and, therefore, staining often fails, and other methods have to be used.

3.2.2. Scanning electron microscopy (SEM) SEM has been used to circumvent this problem and to obtain additional information on the lacunae (Fig. 3) [74,75]. For this the specimen has to be specially prepared so as to be suitable for the necessary vacuum environment during analysis. Typically, cells are first removed, the specimen is dried in an increasing ethanol series and then sputtered with gold to enhance the surface contrast [76]. SEM can be used in combination with stereo-photogrammetry technology to obtain 3-D information on the lacunae [77]. For this purpose stereo-photogrammetry uses images of the pit obtained from different angels to calculate the volume of the pit

Table 2 Typical cell-based resorption lacunae/pit assays. Methods

Measuring parameters

Pros and Cons

References

Light microscopy

Osteoclast number, pit number, pit area

Scanning electron microscopy

Osteoclast number, pit area, pit number

Confocal microscopy

Number of osteoclasts (with specific staining) Pit area, pit depth, pit number, pit volume

Jones et al. [97] Chambers et al. [39] Parikka et al. [85]

Atomic force microscopy

Pit area, pit depth, pit number, pit volume

Super depth surface profile measurement microscopy Infinite focus microscopy

Pit area, pit depth, pit number, pit volume

Pros: availability, cheap Cons: staining necessary Pros: availability Cons: time consuming, labor -intensive, expensive Pros: 3D Cons: fluorescent dye bleaching material specific (collagen) availability, expensive Pros: 3D, no sample processing Cons: availability small detection zone non steep surface Pros: 3D, no sample processing Cons: availability, expensive Pros: 3D, no sample processing, moderate costs Cons: availability

Number of osteoclasts, pit area, pit depth, pit number, pit volume

Bozec et al. [98] Soysa et al. [87] Winkler et al. [29,89]

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Fig. 1. Multinucleated human osteoclasts cultivated from human peripheral blood monocytes after 4 weeks stimulation with RANKL and M-CSF. Light microscopy with TRAP staining (red). Scale bar 50 lm.

Fig. 4. Infinite focus microscopy allows visualization of the depth of the resorption pits in false colours. Infinite focus microscopy. Scale bar 200 lm.

size of the scanning area, which is on the micrometer scale and, therefore, cannot evaluate more than one or two resorption pits in one analysis. In addition, the nature of the detection probe means that it cannot normally be used to measure steep surfaces, which are common in osteoclastic lacunae.

Fig. 2. Osteoclastic pit formation on dentin. Resorption pits are stained blue. Light microscopy with toluidine staining. Scale bar 100 lm.

Fig. 3. Resorption of a calcium phosphate cement by human osteoclasts. The original relatively flat surface is visible in the top left and bottom right corners. Scanning electron microscopy. Scale bar 100 lm.

[78–80]. However, this technology is time consuming, labor intensive and expensive.

3.2.3. Atomic force microscopy (AFM) Another method that was evaluated to analyze resorption lacunae in three dimensions is AFM. AFM is a high resolution microscopy technique with a resolution on the nanometer scale [81]. The surface information is gathered by a cantilever with a sharp tip (probe). Compared with SEM, it can provide real 3-D volumetric data on the sample surface [82]. Furthermore, it can work under ambient conditions without needing a vacuum or sample coating, which may harm the sample surface and affect the results. However, AFM has some limitations. The major limitation is the

3.2.4. Confocal laser scanning microscopy (CLSM) CLSM was invented and commercialized in the 1980s. The principle of confocal microscopy technology is the use of a pinhole to exclude all the out of focus light during the detection phase. The images obtained by confocal microscopy are actually point illustrations [83]. One important step of such a technology is staining of the samples with special fluorescent dyes for illustration [84,85]. Collagen type I is a good target for fluorescent dyes showing collagen fibers within a resorption pit. Combined with F-actin staining with phalloidin, confocal microscopy can show a very clear z-axis resorption pit with the osteoclasts overlaying it [73,86]. The necessary equipment is expensive and the staining procedures are time consuming. Similarly to LM, this technology is also dependent on stainable components, which are not necessarily present in biomaterials. Furthermore, the fluorescent dyes developed so far bleach under laser light. Thus the staining and observation procedure needs to be very accurate to obtain reproducible results. To circumvent the problems associated with the staining procedure an advanced technology based on confocal microscopy technology termed super depth surface profile measurement microscopy (SDSPMM) has been developed [87,88]. No fluorescent dye is needed and the operation of such a system is much easier compared with the conventional confocal microscopy technology. SDSPMM can detect position information via the laser intensity from a target surface point. A 3-D surface map can be visualized as a colored topography and it is possible to quantify this data in three dimensions. 3.2.5. Infinite focus microscopy (IFM) Recently we introduced IFM as a possible tool for measuring bone substitute resorption [29,89] (Fig. 4). The IFM system makes use of the fact that conventional LM has a very narrow focal plane. Software is used that can detect focused areas in images, similar to those that provide the autofocus function in consumer cameras. The observer first takes a series of images (image stack) between the two focal limits. Then the algorithm combines only the focused parts of each image with information on the localization of the image in the image stack. From these data the 3-D surface can be reconstructed at high resolution (<1 lm). No sample preprocessing step is necessary and the size of the detection area is only limited by the computer power [90,91]. Due to its high resolution this method is, however, very susceptible to inaccuracies in the sample geometry. Fitting of the resorption surface plane to the surface of the 3-D dataset and calculating the exclusion plane to account

Z. Zhang et al. / Acta Biomaterialia 8 (2012) 13–19

for the initial roughness of the material are, therefore, necessary steps to exclude artifacts [89]. 3.2.6. Limitations of the current methods Just as for the in vivo assays, cell-based in vitro bone substitute resorption assays have some disadvantages. Due to the low depth of the lacunae and their small size the original roughness of the surface of the material before the assay needs to be very low (ideally Ra < 1 lm) to be able to discriminate between originally existing holes in the surface and newly formed pits. Secondly, none of the above mentioned methods give accurate results when 100% of the surface is resorbed because the measurements will compute the resorbed area and volume relative to the original surface. They will, therefore, need some of the original surface remaining. Moreover, cell-based assays can so far only mimic the resorption of flat bone surfaces, which in vivo are usually only located on the surface of the cortical bone. The greater bone area by far is to be found in the trabecular or spongy bone. A variety of bone substitute biomaterials try to mimic this structure with interconnecting pores, but at present it is nearly impossible to reliably study resorption of such a structure in vitro. 4. Conclusion and outlook The current methods already allow the cell-based analysis of bone biomaterial resorption. There are different cell types, different culture techniques and different analysis methods in use. This leaves the respective scientist with a number of options. From our experience the use of RAW 264.7 cells allows reproducible results with a manageable effort and is, therefore, suitable for a first screening. For results in a human system we suggest differentiating human mononuclear cells to osteoclasts directly on the material, because this models the in vivo situation as closely as possible in vitro. Choice of the right method for analysis of cellular resorption probably largely depends on availability of the necessary equipment. Apart from LM, all the other options are relatively expensive and, therefore, not necessarily accessible to every researcher. At present we would recommend IFM because it gives 3-D results without additional preparation of the specimens, which can always lead to artifacts. As cell cultivation, especially of human cells, is quite demanding and usually takes months until reproducibility can be established, scientists with a focus on material science may want to cooperate with a group experienced in osteoclast cellular biology. Problems with the optical measurements of resorption may possibly be overcome in the future by non-optical X-ray based micro-computed tomography (micro-CT) technology. This technique has already been successfully applied in a whole bone culture assay [92] and to bone resorption in vivo. With computer-assisted 3-D reconstruction the microarchitecture of bone or bone substitutes can be readily identified and quantified [93], and this may even allow resorption monitoring online. Although the skeletal system is commonly perceived to be quite similar in humans the bone structure can vary considerably between subjects [94,95]. Individual patients who suffer from large bone defects may one day profit from the individualized selection of bone biomaterials tailored for their specific needs. Before this distant goal can be achieved several organizational standards will first have to be applied: a commonly accepted standard for evaluation of cell-based material resorption will be needed to make possible the comparison of in vitro results between different groups. The first challenge in this effort will be standardization of the procedure, as cell culture experiments typically show great variability between different laboratories. This problem may be overcome by the definition of a standard material with known

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resorption properties that can be co-cultured as an internal standard [31]. Initially we proposed dentin as such a material, however, this may not be the ideal choice as it is a natural material with uncontrollable natural variations in its properties. It would also be possible to standardize the cell type by using a cell line. At present the only available cell line for resorption assays (RAW 264.7) is of murine origin. The next step then will be to analyze if and how the results of the cell-based assays correspond to the in vivo behavior of the materials. This may eventually lead to a classification of materials based on their resorption behaviors. Only with such a classification will it be possible to evaluate the clinical outcome of grafting procedures based on the different resorption rates of biomaterials. Novel methodologies which allow 3-D reconstructions of micron sized resorption pits literally give us a deeper image of biomaterial resorption. Whether the additional information on the depth of the lacunae will be beneficial in the development of novel materials remains to be seen. In summary, finding the ideal resorbable bone grafting biomaterial for every patient is still a competitive and ongoing task for researchers, clinicians and industry [96]. To manufacture a bone graft substitute with good mechanical properties and good integration in the bone remodeling process will require extensive collaboration between clinicians, physicists, chemists, engineers, biologists and business representatives. Cell-based bone substitute resorption assays may be used for high throughput screening of novel materials and should be considered as part of the preclinical evaluation process for bone substitutes. Description of the human osteoclast differentiation process has made customized bone substitute evaluation possible. In addition, this information can be implemented in bone tissue engineering strategies. With the developments in computer and microscopy technology more detailed cell-based evaluation of bone substitutes can be expected in the future. Hopefully this will lead to an in-depth understanding of cell–material interactions and, consequently, to better materials for our patients. Disclosures In the past five years A.F.S. has provided consulting services to Biomet, Curasan, Eucro, Heraeus and Johnson & Johnson. Acknowledgement The authors would like to thank Mark di Frangia for critical reading of the manuscript. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2 and 4, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio.2011. 09.020. References [1] Teitelbaum SL. Bone resorption by osteoclasts. Science 2000;289:1504. [2] Ross FP, Christiano AM. Nothing but skin and bone. J Clin Invest 2006;116:1140. [3] Onoda S et al. Use of vascularized free fibular head grafts for upper limb oncologic reconstruction. Plast Reconstr Surg 2011;127:1244. [4] Sen MK, Miclau T. Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? Injury 2007;38(Suppl 1):S75. [5] Greenwald AS, Boden SD, Goldberg VM, Khan Y, Laurencin CT, Rosier RN. Bone-graft substitutes: facts, fictions, and applications. J Bone Joint Surg Am 2001;83A(Suppl 2)Pt 2:98. [6] Parikh SN. Bone graft substitutes: past, present, future. J Postgrad Med 2002;48:142.

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