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Osteogenesis induced by a three-dimensional bioimplant composed of demineralised bone matrix, collagen, hydroxyapatite, and bone marrow-derived cells in massive bone defects: An experimental study
Tissue and Cell 50 (2018) 69–78
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Osteogenesis induced by a three-dimensional bioimplant composed of demineralised bone matrix, collagen, hydroxyapatite, and bone marrowderived cells in massive bone defects: An experimental study
T
Carlos E. Cuervo-Lozanoa, Adolfo Soto-Domínguezb, Odila Saucedo-Cárdenasb,c, Roberto Montes-de-Oca-Lunab, Sergio Alonso-Romerod, María del Consuelo Mancías-Guerrae, ⁎ Eduardo Álvarez-Lozanoa, Servicio de Traumatología y Ortopedia, Hospital Universitario “Dr. José Eleuterio González”, Universidad Autónoma de Nuevo León. Av. Madero y Gonzalitos, Monterrey, N.L., A. P. 1563, Mexico Departamento de Histología, Facultad de Medicina, Universidad Autónoma de Nuevo León. Av. Madero y E. Aguirre-Pequeño, Monterrey, N.L., A. P. 1563, Mexico c Centro de Investigación Biomédica del Noreste (CIBIN), IMSS. San Luis y 2 de Abril. Col. Independencia. Monterrey, N.L., C.P. 64720, Mexico d Departamento de Investigación, CIATEC. León, Gto., Mexico e Servicio de Hematología, Hospital Universitario “Dr. José Eleuterio González”, Universidad Autónoma de Nuevo León. Av. Madero y Gonzalitos, Monterrey, N.L., A. P. 1563, Mexico a
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Massive bone defects Osteogenesis Bioimplant Tissue engineering
Treatment of massive bone defects is one of the most difficult problems to solve in orthopedics. At present, there is no consensus on the best way to resolve these problems. The aim of our study was to evaluate the effect of a three-dimensional bioimplant over massive bone defects, and to analyse if it improves the speed and quality of integration in recipient bone compared to allograft treatment. Fifteen female lambs with massive bone defects, surgically created in their tibias, were randomly divided into three groups of five lambs each: Group I −treated with the bioimplant; Group 2 −treated with the bioimplant plus nucleated cells of autologous bone marrow; Group 3 −treated with a frozen allograft. Radiographs were taken post-treatment at weeks 1, 6, and 12. Animals were euthanized to obtain the studied bone segment for morphological analyses. Treatment: with bioimplants vs. bioimplant plus bone marrow nucleated cells (BMNCs) showed a notorious osteogenic effect, but with greater osteoid synthesis and cellularity in the latter. These results suggest that combined treatment with bioimplants and BMNCs have an additive effect on massive bone defects in lambs. These experimental results could be applied to repair damaged human bone.
1. Introduction
shape, and size. Additionally, the patient undergoes a second surgical procedure, thus increasing morbidity by 10% (Younger and Chapman, 1989). The use of allografts has increased in the last 20 years, but these are also far from ideal because of the lack of complete integration, especially for large defects (Bus et al., 2014); this problem leads to its main complications, such as fractures, infections, and resorptions (Ayvaz et al., 2014; Delloye et al., 2014; San-Julian and Canadell, 1998). The recent development of “tissue engineering” offers new options for repairing bone defects (Langer and Vacanti, 1993) and one of the corresponding fields is the creation of three-dimensional bioimplants that provide a suitable environmental medium for tissue growth (Cunniffe et al., 2010; Hansen et al., 2005). Demineralised bone matrix
Since the beginning of the 1970s, when the era of limb salvage began (Eilber et al., 1980), different forms of reconstruction have been used to treat massive bone defects, ranging from the use of an autograft, allografts (Aponte-Tinao et al., 2014, 2015, 2016; Houdek et al., 2016; Rabitsch et al., 2013), bone substitutes, special prostheses (Gkavardina and Tsagozis, 2014; Tiwari et al., 2014), or combinations of these reconstruction methods (Antoci et al., 2009; Arslan et al., 2015; Qu et al., 2015; Sevelda et al., 2015; Venkatramani et al., 2015; Vlad et al., 2013). Autologous bone remains the gold standard for bone grafts; however, it has the enormous disadvantage of being limited in quantity,
⁎ Corresponding author at: Servicio de Traumatología y Ortopedia, Hospital Universitario "Dr. José Eleuterio González", Universidad Autónoma de Nuevo León, Av. Madero y Gonzalitos S/N Colonia Mitras Centro, Monterrey, N.L., México. A. P.1563. E-mail address: [email protected] (E. Álvarez-Lozano).
https://doi.org/10.1016/j.tice.2017.12.005 Received 23 September 2017; Received in revised form 7 December 2017; Accepted 9 December 2017 Available online 12 December 2017 0040-8166/ © 2017 Elsevier Ltd. All rights reserved.
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[DBM] (Urist, 1965; Vaccaro et al., 2002) is a very useful material because it has bone morphogenetic proteins [BMPs] (Urist et al., 1970; Urist and States, 1971), which act as osteoinductive mediators; however, an osteoinductive agent without a carrier will be lost or degraded rapidly without producing any osteogenic effect (Duflo et al., 2006; Hotz and Herr, 1994; Liu et al., 2006). Because bone is a nanostructured tissue (Balasundaram and Webster, 2007), this carrier must have certain chemical and structural characteristics (Seref-Ferlengez et al., 2015; Song et al., 2015; Terauchi et al., 2015). “Stem cells” (Nowakowski et al., 2015) have recently aroused a growing interest in their therapeutic potential in diseases that until now lacked an effective treatment. These cells are found in the quiescent state in the body, mainly in bone marrow and adipose tissue, as multipotent stromal cells (Maumus et al., 2011; O'Brien et al., 2007). The structure, cellularity, function, and biological mechanisms of bone tissue make it difficult to find a material, biological or otherwise, that replaces bone and replicates all its characteristics. It is important to provide a biological and structural aid to the body to facilitate the repair of massive bone defects through fast, high-quality integration. The aim of this study was to evaluate if a three-dimensional bioimplant improves the speed and quality of integration in the recipient bones, compared to allograft treatment, of massive bone defects. This bioimplant was generated from the combination of the components of bone tissue: DBM with BMPs, collagen, hydroxyapatite [HAp] Ca10(PO4)6(OH)2, and osteoprogenitor cells.
proximal diaphysis of the left tibia of the lambs using an oscillating saw and a metallic guide specifically fabricated for this purpose. The bone defect comprised 75% of the cortical circumference and 5 cm of the length. The bone defects were treated in three different manners; animals were randomly selected and divided into 3 groups of five lambs each. Group one lambs were treated only with the bioimplant. Group two lambs were treated with the bioimplant plus autologous nucleated cells; prior to its placement, the bioimplant was immersed for 5 min in a container with the cells obtained from an iliac crest aspirate by a special centrifugation process a few hours before surgery. In group three, the defect was treated with a frozen allograft. In all cases, internal fixation with a special plate and screws was used to keep the allograft in place. The lambs remained in the bioterium for 5 days for wound monitoring and administration of antibiotic (20 mIU procaine benzylpenicillin) without restriction of limb support and with ad libitum access to water and food. From the 6th day onwards, the animals were transferred to a common habitat. The experiments were performed in accordance with the International Guidelines on the Appropriate Use of Experimental Animals and according to the Mexican Norm NOM-062-ZOO-1999 on the Technical Specifications for the Production, Care and Use of Laboratory Animals (SAGARPA, 1999). The protocol was approved by the Bioethical Committee of the Faculty of Medicine, UANL in Monterrey, Nuevo León, México.
2. Materials and methods
2.4. Radiological analysis
2.1. Study design and setting This is an experimental, longitudinal, comparative, blind and controlled study.
In the radiological analysis, antero-posterior and lateral radiographs of the limb were taken at weeks 1 (control), 6, and 12 post-treatment and the appearance of the massive bone defects in the tibias of the study groups were evaluated.
2.2. Preparation of bioimplant
2.5. Histological and histochemical analysis
The first component used for the bioimplant was DBM, which was generated via the demineralisation of the diaphysis of the long bones of 3 hind limbs of donor lambs using a technique previously described by Rivera et al. (2003). The DBM has biological characteristics that favour osteoinduction (Urist et al., 1970; Urist and Strates, 1971; Vaccaro et al., 2002). The second component was collagen because collagen is the most abundant structural protein in bone; collagen was used to provide rigidity and maintain the desired shape of the bioimplant while making the implant insoluble when contacting body fluids. Collagen was obtained from the skin of foetal pigs by a process consisting of mechanically macerating the skin in a meat grinder and dripping a 0.5% by weight solution of hydrochloric acid [HCl] with constant stirring in distilled water. Bone is a nanostructured tissue, and therefore to achieve a porosity of less than 100 nm, HAp nanoparticles were selected as the third component. HAp is the main inorganic component of bone and its use in nanoparticles improves the microenvironment and increases the useful surface area for cell growth. The HAp nanoparticles were processed from waste egg shells by a hydrothermal synthesis process, as described in the technique published by Elizondo-Villarreal et al. (2012). The three components were mixed in a proportion of 30% DBM, 60% collagen, and 10% HAp. The mixture was placed in prefabricated 5-cc moulds of a specific shape and size, dried, and subsequently sterilised in doses of 20–22 kg.
Animals euthanized with sodium pentobarbital (90–210 mg/kg weight, intravenous administration) at 12 weeks to obtain the studied bone segment for morphological analyses. After the samples were collected, they were fixed in 4% paraformaldehyde solution in 1X phosphate buffered saline [PBS] pH 7.2–7.4 for 24 h. Subsequently, a 1-cmthick segment was collected and treated with the 10% HCl decalcification technique for 21 days by changing the solution every third day, observing that the bone showed a soft consistency. Afterward, samples were processed by conventional histological techniques until their inclusion in paraffin blocks. The general cellular characteristics, presence of bone trabeculae, and orientation and organisation of the collagen fibres were evaluated in histological sections (4 μm thick) stained with haematoxylin and eosin [H&E] and Mallory-Azán Trichrome [M-AT] for histological analysis. Additionally, the histochemical periodic acid-Schiff [PAS] staining method was used to identify the components of the osteoid. Samples were evaluated using light field microscopy. 2.6. Immunohistochemical analysis To identify the presence of osteoblasts in the samples of interest, immunohistochemical analysis was performed in 4-μm-thick histological sections immunolabeled with polyclonal anti-Parathyroid Hormone Receptor R1[PTHR/PTHR1] antibody (aa388-406, LSC313515 (1:200), LifeSpan Biosciences, Inc., Seattle, WA, USA). An Abcam® anti-mouse & rabbit HRP/DAB detection kit (ab64264 Cambridge, MA, USA) was used as the detection system. Positivity was visualised with 3,3′-diaminobenzidine [DAB] and the nuclei were identified using Gill’s haematoxylin.
2.3. Description of experiment, treatment, or surgery We employed 15 female lambs (Ovis aries), 4–5 months of age and weighing 20–30 kg. A bone defect was surgically created in the 70
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weeks 1 (control), 6, and 12 post-treatment for radiological evaluation. The radiographs were evaluated by 3 different orthopaedic doctors and they gave 1 point if the x-ray showed no ossification or integration, 2 points if the ossification or integration was less than 50%, and 3 points if they found more than 50% ossification or integration. The anteroposterior radiograph in the first postoperative week with a bioimplant plus the nucleated cells of autologous bone marrow showed the bone defect (Fig. 1E). At week 12, the same limbs showed 100% ossification (Fig. 1F). We didńt find any statistical difference between the groups.
2.7. Morphometric analysis To quantify the osteoid percentage, the samples were grouped according to the type of treatment without specifying the treatment; 40 consecutive fields were photodocumented in each group with a strong dry objective (40×) in the PAS-positive samples (8 fields/slice, 1 slice/ lamb, and 5 lambs/group). High-resolution digital images were obtained with a Nikon Eclipse 50i microscope and a Digital Sight dDS2Mu image analysis system with NIS-Elements software. Subsequently, the images were analysed with ImageJ version 1.49 (National Institutes of Health); colour, tone distribution, saturation, and illumination were the same for all the images. This morphometric analysis allowed us to determine the osteoid percentage in the study groups. Subsequently, in these same images, the intensity [IntDent]of the positivity in the osteoid in each sample was quantified by microdensitometry. The values obtained were expressed as optical density. The mean value and the standard deviation [SD] of all the values from the morphometric analyses were obtained for statistical analysis and for comparison between the study groups. In addition, morphometric analysis of PTHR/PTHR1-positive cells was performed in which the positive cells/field were quantified in 40 fields for each group with the same methodology described previously. The morphological analysis of the samples was performed by two specialists in morphology who were blinded to the treatment used in each sample and blinded to each other.
3.3. Histological analysis In the histological analysis of the group 1 implantation site (bioimplant), we observed areas with different collagen fibre organisation and cell layout, so they were classified into three areas according to the cellular and tissue organisation (Fig. 2A). Zone 1 showed irregularly organised fibres with cells between and at the periphery of the bundles; notably, in this zone, the spaces are similar to small blood vessels between the collagen fibres (Fig. 2D). In zone 2, a larger organisation of the fibres and cells was observed, as well as larger-diameter spaces (Fig. 2G). Finally, in zone 3, the histological characteristics of an immature bone tissue are clearly observed: bundles of collagen fibres, which began to organise around a cavity with cells in its interior, similar to the endosteum of the Haversian canals. In addition, round cells similar to osteocytes were observed in the gaps between the fibres (Fig. 2J). An important finding in this group was the presence of areas of acidophilic material that represent osteoid (Fig. 2D). In group 2 (bioimplant+ bone marrow nucleated cells), the same previously-described areas were observed (Fig. 2B). Notably, a higher cellularity was observed in this group compared to group 1 (Fig. 2E). Moreover, in this group, the organisation of osteon-like structures was greater than in group 1 (Fig. 2H and K) and a greater amount of osteoid was observed (Fig. 2E). Finally, in group 3 (allograft), clear divisions were observed between the bone tissue laminae with a distinct organisation of the osteons (Fig. 2C). In this group, endosteal remnants were observed in the Haversian canals (Fig. 2F and I) and there were no cells in the gaps of the concentric laminae (Fig. 2L). The previously-described results were corroborated with the M-AT stain. Pale red lines indicative of an osteoid were observed in the
3. Results 3.1. Characterisation of the implant Prior to implantation, the bioimplant was characterised; we observed collagen fibre bundles with acidophilic staining and cells with basophilic nuclei (Fig. 1A and B). These data were confirmed by the MAT stain showing the blue collagen fibres and cells with red cytoplasm (Fig. 1C). Also, in the bioimplant samples, we observed small, semitransparent granular structures corresponding to the DBM and HAp (Fig. 1D). 3.2. Radiological analysis Antero-posterior and lateral radiographs of the limbs were taken at
Fig. 1. Morphological characterisation of bioimplant: A and C) Panoramic images showing bundles of collagen fibres. B and D) Amplification of the boxes showing the collagen fibres (red arrows) and demineralised bone matrix (yellow arrow). Light microscopy. Inclusion in paraffin. A and B: H&E stain; C and D: M-AT stain. E and F): Antero-posterior X-ray of lamb tibia, 1 week (E) and 12 weeks (F) after bone defect treated with bioimplant plus nucleated cells from bone marrow, showing 100% ossification. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. The use of bioimplant has an osteogenic effect in vivo: A, B and C) Panoramic images of group 1, group 2, and group 3. D and G) Amplifications of boxes 1 and 2 of image A, collagen fibre bundles (blue arrows) with cells between the bundles (yellow arrows) and at the periphery (white arrows), and small vessels (green arrows). J) Amplification of box 3 of image A corresponding to zone 3, which shows osteons with concentric laminae (blue arrow) around blood vessels (green arrow) and with osteocytes in their lagoons (yellow arrow). E and H) Amplifications of boxes 1 and 2 of image B, with the same elements indicated for group 1. K) Amplification of box 3 of image B corresponding to zone 3.There is a greater organisation of osteons with concentric sheets (blue arrow) around areas resembling small blood vessels (green arrow) and with osteocytes in their lagoons (yellow arrow) than in group 1. F, I and L) Amplifications of boxes 1, 2, and 3 of image C, plaques of bone tissue (blue arrows), with spaces without cells (white arrows) and endosteal remnants in the Havers canals (green arrows). Light microscopy. Inclusion in paraffin. H&E stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Histochemical analysis
periphery of the developing bone trabeculae or in areas that contained cells in groups 1 and 2 with this staining (Fig. 3A, D, G and J); this feature was more pronounced in group 2 (Fig. 3B, E, H and K). On the other hand, in group 3, few areas with the previously described characteristics of an osteoid were observed via this staining (Fig. 3C, F, I and L).
In addition, this method permitted the identification of positivity in the cells of both group 1 (Fig. 4 and D) and group 2 (Fig. 4B and E). It should be noted that this stain allowed the identification of extensive areas with positivity in developing bone trabeculae as well as areas with amorphous PAS material suggestive of extracellular matrix 72
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Fig. 3. Bioimplant induces osteoid secretion: A, B and C) Panoramic images of study groups. D) Amplification of box 1 of image A, collagen fibre bundles (green arrow) with cells (yellow arrow). G and J) In zones 2 and 3, there is a greater organisation of fibres similar to osteons with concentric laminae (green arrows), cells (yellow arrows), and pale red lines indicative of an osteoid in the periphery of developing bone trabeculae or in areas that contained cells in red (red arrows). E and H) Amplifications of boxes 1, 2 and 3 of image B, with the same elements indicated for group 1. K) There is a greater organisation of osteons with concentric laminae (green arrow), there are osteocytes in their lagoons (yellow arrow), and osteoid staining was more pronounced (red arrow). F, I and L) Amplifications of boxes 1, 2 and 3 of image C, plaques of bone tissue (green arrow), with spaces without cells (yellow arrows) and few areas with osteoid characteristics (red arrow). Light microscopy. Inclusion in paraffin.M-AT stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
program, the following values were observed: 4.429E + 07 ± 3.135E + 07 for group 1, 1.554E + 08 ± 4.07E + 07 for group 2, and 4.14E + 06 ± 2.15E + 06 for group 3. As shown in the graph, group 2 showed the highest intensity, followed by group 1 and finally group 3 (Fig. 5B).
synthesised by the cellular elements (Fig. 4G, J, H, and K). Interestingly, greater positivity was observed in group 2 than in groups 1 and 3, with the latter showing few areas of positivity (Figs. 4C, F, I, and L). In the morphometric analysis of PAS histochemical positivity in the osteoid of each of the study groups, values of 5.831% ± 2.240% were observed in group 1, 16.585% ± 3.597 in group 2, and 1.316% ± 0.865 in group 3 (Fig. 5A). When the intensity of the density was quantified using the ImageJ 73
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Fig. 4. The use of bioimplant activates osteoid synthesis in vivo: A, B and C) Panoramic images of groups. D, G and J) Amplifications of zones 1, 2 and 3 of image A, cells with positivity in the cytoplasm (green arrows) and positive material between fibres and trabeculae (blue arrow). E, H and K) Amplifications of boxes 1, 2 and 3 of image B, with the same elements identified for group 1. Greater positivity was observed in group 2. F, I and L) Amplifications of the boxes 1, 2 and 3 of image C, thin lines of osteoid (blue arrows). Light microscopy. Inclusion in paraffin. PAS stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In the morphometric analysis of the positive cells, values of 8.175 ± 0.734 were observed for group 1, 11.475 ± 0.531 for group 2, and 1.175 ± 0.090 for group 3. These results demonstrate that the number of osteoblasts was highest in group 2 (Fig. 6G).
3.5. Immunohistochemical analysis The immunolabeling with anti-PTHR/PTHR1-specific antibodies showed areas with positive cells, indicating the presence of osteoblasts; these cells were observed mainly in the samples of groups 1 and 2. Positivity was observed in cells situated around the trabeculae, between the collagen fibres, and in the interior of the spaces, suggestive of vessels or Haversian canals (Fig. 6A–D). Group 3 showed few positive cells (Fig. 6E and F). The bioimplant showed negative results for osteoblast cells (data not shown).
3.6. Statistical analysis, study size The Kolmogorov-Smirnov test showed that the samples were not normally distributed; therefore, we used the Kruskal-Wallis test to compare the groups. A post-hoc test was used and we found a statistical 74
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Fig. 5. Bioimplant shows an increased quantity of osteoid synthesis: A) Morphometric analysis of the osteoid of each of the study groups. Group 2 shows a higher percentage of osteoid than the other groups. B). Morphometric analysis of density of osteoid. Group 2 showed the highest density, followed by group 1 and finally group 3. Statistical differences were found betweenthe groups. * p ≤ 0.05.
may be occurring in this model’s process of regeneration that results in the formation of bone tissue. On the other hand, a greater amount of concentric laminae with spaces similar to Havers' canals characteristic of the osteons of bone tissue was observed in group 2, compared to groups 1 and 3. These results suggest that the use of HAp promotes the organisation of the fibres and cells. Studies with materials for bone repair using an aggregate of mineral trioxide combined with monoolein gel demonstrate the synthesis of bone tissue in defects caused in the jaws of experimental animals (Falcão-Filho et al., 2007). The samples analysed with the M-AT stain had the histological characteristics of immature bone tissue. These results agree with those described for bone-remodelling units [BRUs], including osteoblasts. In the areas with BRUs activity, the osteoid synthesis is observed as red lines on the trabeculae, which were identified with trichrome methods (Velásquez-Forero, 2009). The cells of groups 1 and 2 were PAS-positive in the cytoplasm and there were areas of PAS-positive material with and without the presence of cells. These results suggest that the cells present in the area of the bioimplant synthesise and secrete components of the extracellular matrix. In addition, DBM proteins have been reported to act as modulators of growth factors (Young, 2003). Because the matrix proteins
significance between all groups, group 1 vs group 2, group 1 vs group 3, and group 2 vs group 3.
4. Discussion The results of the present study demonstrate for the first time that implantation of a biomaterial that does not have a specific structure, similar to our bioimplant, in a bone repair model has an osteogenic effect; promoting the synthesis of extracellular matrix and the organisation of collagen fibres in concentric layers around vascular spaces, similar to the histological characteristics of bone tissue. These characteristics were observed after only 3 months of bioimplantation in the in vivo model. In this study, samples of groups 1 and 2 showed large areas with eosinophilic material corresponding to osteoid, with and without the presence of cells around and between the trabeculae of the bone tissue in formation. These findings are consistent with those observed in studies that describe osteogenic events during embryogenesis and later in bone regeneration: cellular aggregates that subsequently differentiate from precursor cells into an osteoblastic lineage, which then deposit and mineralise the osteoid and finally differentiate from osteoblasts to osteocytes (Kaul et al., 2015). Therefore, these phenomena 75
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Fig. 6. Bioimplant shows an increased number of osteoblasts in vivo: A-F) Immunolabeling with anti-PTHR/PTHR1-specific antibodies in groups 1, 2 and 3.Areas with positive cells indicating the presence of osteoblasts (yellow arrows) and negative cells (green arrows). These cells were mainly observed in the samples of groups 1 (A and B) and 2 (C and D). Group 3 showed few positive cells (E and F). Light microscopy. Inclusion in paraffin. Immunohistochemistry. G) Quantification of positive cells/field in each study group. An increased number of positive cells were observed in group 2. Statistical differences were found between the groups. * p ≤ 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
bioimplant. The morphogenic proteins are abundant in bone tissue, and we know that BMPs are involved in the formation of bone and cartilage during embryogenesis (Fernandez-Tresguerres-Hernandez-Gil et al., 2006). Also, BMPs are now considered one the most potent factors of osteoblastic differentiation (Yamaguchi et al., 2000). Canalis et al. reported that in addition to stimulating osteogenesis, morphogenic proteins inhibit osteoclasto genesis (Canalis et al., 2003). Furthermore, the induction of bone regeneration has been described in a model of mandibular distraction surgery treated with transduced mesenchymal stem cells with the human morphogenic protein-2
are at a concentration 1000 times greater than the growth factors, these proteins could play a more important role in the regulation of different cellular functions (Horowitz, 2003). We did not find reports in the literature that described these phenomena that are induced by the components of the bioimplant used in this study. Finally, some cells were identified by immunolabeling with antiPTHR1-specific antibodies as osteoblasts. These results suggest that the use of our bioimplant induces the differentiation and/or migration of cells. In this study we used the DBM of lamb with BMPs as part of the
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(Castro-Govea et al., 2012). Moreover, BMPs also induce the differentiation of osteoprogenitor cells and participate in the regulation of osteoblastic cell proliferation, migration, and differentiation into osteocytes (Fernandez-TresguerresHernandez-Gil et al., 2006). To date, the mechanisms of cell differentiation and stimulation of osteoid synthesis observed in this study remain unknown; however, these results will be useful for the development of new therapeutic strategies that seek to shorten the time of bone repair and to obtain a greater organisation of the cellular and fibrous elements present in the bone tissue.
Balasundaram, G., Webster, T.J., 2007. An overview of nano-polymers for orthopedic applications. Macromol. Biosci. 7, 635–642. Bus, M.P., Dijkstra, P.D., van de Sande, M.A., Taminiau, A.H., Schreuder, H.W., Jutte, P.C., van der Geest, I.C., Schaap, G.R., Bramer, J.A., 2014. Intercalary allograft reconstructions following resection of primary bone tumors: a nationwide multicenter study. J. Bone Joint Surg. Am. 96, e26. Canalis, E., Economides, A.N., Gazzerro, E., 2003. Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr. Rev. 24, 218–235. Castro-Govea, Y., Cervantes-Kardasch, V.H., Borrego-Soto, G., Martínez-Rodríguez, H.G., Espinoza-Juárez, M., Romero-Díaz, V., Marino-Martínez, I.A., Robles-Zamora, A., Álvarez-Lozano, E., Padilla-Rivas, G.R., 2012. Human bone morphogenetic protein 2–transduced mesenchymal stem cells improve bone regeneration in a model of mandible distraction surgery. J. Craniofac. Surg. 23, 392–396. Cunniffe, G.M., Dickson, G.R., Partap, S., Stanton, K.T., O’Brien, F.J., 2010. Development and characterization of a collagen nano-hydroxyapatite composite scaffold for bone tissue engineering. J. Mater. Sci. Mater. Med. 21, 2293–2298. Delloye, C., van Cauter, M., Dufrane, D., Francq, B.G., Docquier, P.L., Cornu, O., 2014. Local complications of massive bone allografts: an appraisal of their prevalence in 128 patients. Acta Orthop. Belg. 80, 196–204. Duflo, S., Thibeault, S.L., Li, W., Shu, X.Z., Prestwich, G.D., 2006. Vocal fold tissue repair in vivo using a synthetic extracellular matrix. Tissue Eng. 12, 2171–2180. Eilber, F.R., Mirra, J.J., Grant, T.T., Weisenburger, T., Morton, D.L., 1980. Is amputation necessary for sarcomas? A seven-year experience with limb salvage. Ann. Surg. 192, 431–438. Elizondo-Villarreal, N., Martínez-de-la-Cruz, A., Guerra, R.O., Gómez-Ortega, J.L., TorresMartínez, L.M., Castaño, V.M., 2012. Biomaterials from agricultural waste: eggshellbased hydroxyapatite. Water Air Soil Pollut. 223, 3643–3646. Falcão-Filho, H.B.L., Issa, J.P.M., Nascimento, C., Iyomasa, M.M., Regalo, S.C.H., Siéssere, S., Pedrazzi, V., Pitol, D.L., 2007. Histological evaluation of the bone repair using mineral trioxide aggregate combined to a material carrier. Int. J. Morphol. 25 (4), 789–796. Fernandez-Tresguerres-Hernandez-Gil, I., Alobera-Gracia, M.A., del-Canto-Pingarron, M., Blanco-Jerez, L., 2006. Physiological bases of bone regeneration II. The remodeling process. Med. Oral Patol. Oral Cir. Bucal 11, E151–E157. Gkavardina, A., Tsagozis, P., 2014. The use of megaprostheses for reconstruction of large skeletal defects in the extremities: a critical review. Open Orthop. J. 8, 384–389. Hansen, J.K., Thibeault, S.L., Walsh, J.F., Shu, X.Z., Prestwich, G.D., 2005. In vivo engineering of the vocal fold extracellular matrix with injectable hyaluronic acid hydrogels: early effects on tissue repair and biomechanics in a rabbit model. Ann. Otol. Rhinol. Laryngol. 114, 662–670. Horowitz, M., 2003. Matrix proteins versus cytokines in the regulation of osteoblasts function and bone formation. Calcif. Tissue Int. 72, 5–7. Hotz, G., Herr, G., 1994. Bone substitute with osteoinductive biomaterials–current and future clinical applications. Int. J. Oral Maxillofac. Surg. 23, 413–417. Houdek, M.T., Wagner, E.R., Stans, A.A., Shin, A.Y., Bishop, A.T., Sim, F.H., Moran, S.L., 2016. What is the outcome of allograft and intramedullary free fibula (Capanna technique) in pediatric and adolescent patients with bone tumors? Clin. Orthop. Relat. Res. 474, 660–668. Kaul, H., Hall, B.K., Newby, C., Ventikos, Y., 2015. Synergistic activity of polarised osteoblasts inside condensations cause their differentiation. Sci. Rep. 5, 11838. Langer, R., Vacanti, J.P., 1993. Tissue engineering. Science 260, 920–926. Liu, Y., Ahmad, S., Shu, X.Z., Sanders, R.K., Kopesec, S.A., Prestwich, G.D., 2006. Accelerated repair of cortical bone defects using a synthetic extracellular matrix to deliver human demineralized bone matrix. J. Orthop. Res. 24, 1454–1462. Maumus, M., Peyrafitte, J.A., D'Angelo, R., Fournier-Wirth, C., Bouloumie, A., Casteilla, L., Sengenes, C., Bourin, P., 2011. Native human adipose stromal cells: localization, morphology and phenotype. Int. J. Obes. 35, 1141–1153. Nowakowski, A., Walczak, P., Janowski, M., Lukomska, B., 2015. Genetic engineering of mesenchymal stem cells for regenerative medicine. Stem Cells Dev. 24, 2219–2242. O'Brien, F.J., Harley, B.A., Waller, M.A., Yannas, I.V., Gibson, L.J., Prendergast, P.J., 2007. The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering. Technol. Health Care 15, 3–17. Qu, H., Guo, W., Yang, R., Tang, X., Yan, T., Li, D., Yang, Y., Zang, J., 2015. Cortical strut bone grafting and long-stem endoprosthetic reconstruction following massive bone tumour resection in the lower limb. Bone Joint J. 97B, 544–549. Rabitsch, K., Maurer-Ertl, W., Pirker-Frühauf, U., Wibmer, C., Leithner, A., 2013. Intercalary reconstructions with vascularised fibula and allograft after tumour resection in the lower limb. Sarcoma 2013, 8. Rivera, J.A., Benavides, C.R., Monsalve, P.A., Injertos, Osorio A., 2003. óseos-Nueva alternativa: Fase I. Extracción de proteínas morfogenéticas parcialmente purificadas de hueso bovino. Rev. Colomb. Cienc. Pec. 16, 139–146. San-Julian, M., Canadell, J., 1998. Fractures of allografts used in limb preserving operations. Int. Orthop. 22, 32–36. Seref-Ferlengez, Z., Kennedy, O.D., Schaffler, M.B., 2015. Bone microdamage, remodeling and bone fragility: how much damage is too much damage? Bonekey Rep. 4, 644. Sevelda, F., Schuh, R., Hofstaetter, J.G., Schinhan, M., Windhager, R., Funovics, P.T., 2015. Total femur replacement after tumor resection: limb salvage usually achieved but complications and failures are common. Clin. Orthop. Relat. Res. 473, 2079–2087. Song, W., Li, X., Qian, J., Lv, G., Yan, Y., Su, J., Wei, J., 2015. Mesoporous calcium-silicon xerogels with mesopore size and pore volume influence hMSC behaviors by load and sustained release of rhBMP-2. Int. J. Nanomed. 10, 1715–1726. Terauchi, M., Ikeda, G., Nishida, K., Tamura, A., Yamaguchi, S., Harada, K., Yui, N., 2015. Supramolecular polyelectrolyte complexes of bone morphogenetic protein-2 with sulfonated polyrotaxanes to induce enhanced osteogenic differentiation. Macromol. Biosci. 15, 953–964.
5. Conclusions The results observed in this study allow us to propose the use of our bioimplant with autologous nucleated bone marrow cells for bone repair because this combination showed a stronger osteogenic effect in the in vivo model, as analysed by morphological and morphometric tests, than did the cell-free bioimplant or the use of an allograft. 6. Background and rationale At the present time there is no material, biological tissue or otherwise, with the structure, cellularity, function, and biological mechanisms of bone tissue. We are not trying to find “the magic tissue” that replaces bone and replicates all its characteristics. Our goal is to provide the body with a biological and structural aid to facilitate the repair of massive bone defects while producing higher-quality repairs that require less time to heal. 7. Limitations The main limitation of this study was that we did not use flow cytometry to analyse cells obtained from an iliac crest aspirate and name them “stem cells”; instead, we named them autologous nucleated cells from iliac crest, even knowing that many of these cells are “stem cells”. Conflicts of interest None. Acknowledgements We thank Federico A. Rodríguez Ph.D. and Nora Elizondo Ph.D. for their investigation in biomaterials and donation of nanoparticles, and Jorge Lozano for his contribution regarding the investigation animals.We also thank Luis I. Botello S. and Iván A. Mesta R. for technical assistance involved in processing the samples for morphological analysis. References Antoci, V., Phillips, M.J., Antoci Jr., V., Krackow, K.A., 2009. Using an antibiotic-impregnated cement rod-spacer in the treatment of infected total knee arthroplasty. Am. J. Orthop. 38, 31–33. Aponte-Tinao, L.A., Ritacco, L.E., Albergo, J.I., Ayerza, M.A., Muscolo, D.L., Farfalli, G.L., 2014. The principles and applications of fresh frozen allografts to bone and joint reconstruction. Orthop. Clin. North Am. 45, 257–269. Aponte-Tinao, L., Ayerza, M.A., Muscolo, D.L., Farfalli, G.L., 2015. Survival, recurrence, and function after epiphyseal preservation and allograft reconstruction in osteosarcoma of the knee. Clin. Orthop. Relat. Res. 473, 1789–1796. Aponte-Tinao, L.A., Ayerza, M.A., Muscolo, D.L., Farfalli, G.L., 2016. What are the risk factors and management options for infection after reconstruction with massive bone allografts? Clin. Orthop. Relat. Res. 474, 669–673. Arslan, H., Ozkul, E., Gem, M., Alemdar, C., Sahin, I., Kisin, B., 2015. Segmental bone loss in pediatric lower extremity fractures: indications and results of bone transport. J. Pediatr. Orthop. 35, e8–e12. Ayvaz, M., Bekmez, S., Mermerkaya, M.U., Caglar, O., Acaroglu, E., Tokgozoglu, A.M., 2014. Long-term results of reconstruction with pelvic allografts after wide resection of pelvic sarcomas. Sci. World J. 2014, 6.
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Venkatramani, H., Sabapathy, S.R., Dheenadayalan, J., Devendra, A., Rajasekaran, S., 2015. Reconstruction of post-traumatic long segment bone defects of the lower end of the femur by free vascularized fibula combined with allograft (modified Capanna's technique). Eur. J. Trauma Emerg. Surg. 41, 17–24. Vlad, C., Gavriliu, T., Georgescu, I., Dan, D., Parvan, A., Burnei, G., 2013. Bone transport with the lengthening through the physis in patients having congenital pseudarthrosis of tibia – short-term results. J. Med. Life. 6, 266–271. Yamaguchi, A., Komori, T., Suda, T., 2000. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr. Rev. 21, 393–411. Young, M.F., 2003. Bone matrix proteins: more than markers. Calcif. Tissue Int. 72, 2–4. Younger, E.M., Chapman, M.W., 1989. Morbidity at bone graft donor sites. J. Orthop. Trauma 3, 192–195.
Tiwari, A., Jain, S., Mehta, S., Kumar, R., Kapoor, G., Kumar, K., 2014. Limb salvage surgery for osteosarcoma: early results in Indian patients. Indian J. Orthop. 48, 266–272. Urist, M.R., Strates, B.S., 1971. Bone morphogenetic protein. J. Dent. Res. 50, 1392–1406. Urist, M.R., Jurist Jr., J.M., Dubuc, F.L., Strates, B.S., 1970. Quantitation of new bone formation in intramuscular implants of bone matrix in rabbits. Clin. Orthop. Relat. Res. 68, 279–293. Urist, M.R., 1965. Bone: formation by autoinduction. Science 150, 893–899. Vaccaro, A.R., Chiba, K., Heller, J.G., Patel, T., Thalgott, J.S., Truumees, E., Fischgrund, J.S., Craig, M.R., Berta, S.C., Wang, J.C., 2002. Bone grafting alternatives in spinal surgery. Spine J. 2, 206–215. Velásquez-Forero, F.H., 2009. Histomorfometría de la biopsia ósea previo marcaje y procesada sin descalcificar. Patología 47, 108–117.
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Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Osteogenesis induced by a three-dimensional bioimplant composed of demineralised bone matrix, collagen, hydroxyapatite, and bone marrowderived cells in massive bone defects: An experimental study
T
Carlos E. Cuervo-Lozanoa, Adolfo Soto-Domínguezb, Odila Saucedo-Cárdenasb,c, Roberto Montes-de-Oca-Lunab, Sergio Alonso-Romerod, María del Consuelo Mancías-Guerrae, ⁎ Eduardo Álvarez-Lozanoa, Servicio de Traumatología y Ortopedia, Hospital Universitario “Dr. José Eleuterio González”, Universidad Autónoma de Nuevo León. Av. Madero y Gonzalitos, Monterrey, N.L., A. P. 1563, Mexico Departamento de Histología, Facultad de Medicina, Universidad Autónoma de Nuevo León. Av. Madero y E. Aguirre-Pequeño, Monterrey, N.L., A. P. 1563, Mexico c Centro de Investigación Biomédica del Noreste (CIBIN), IMSS. San Luis y 2 de Abril. Col. Independencia. Monterrey, N.L., C.P. 64720, Mexico d Departamento de Investigación, CIATEC. León, Gto., Mexico e Servicio de Hematología, Hospital Universitario “Dr. José Eleuterio González”, Universidad Autónoma de Nuevo León. Av. Madero y Gonzalitos, Monterrey, N.L., A. P. 1563, Mexico a
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Massive bone defects Osteogenesis Bioimplant Tissue engineering
Treatment of massive bone defects is one of the most difficult problems to solve in orthopedics. At present, there is no consensus on the best way to resolve these problems. The aim of our study was to evaluate the effect of a three-dimensional bioimplant over massive bone defects, and to analyse if it improves the speed and quality of integration in recipient bone compared to allograft treatment. Fifteen female lambs with massive bone defects, surgically created in their tibias, were randomly divided into three groups of five lambs each: Group I −treated with the bioimplant; Group 2 −treated with the bioimplant plus nucleated cells of autologous bone marrow; Group 3 −treated with a frozen allograft. Radiographs were taken post-treatment at weeks 1, 6, and 12. Animals were euthanized to obtain the studied bone segment for morphological analyses. Treatment: with bioimplants vs. bioimplant plus bone marrow nucleated cells (BMNCs) showed a notorious osteogenic effect, but with greater osteoid synthesis and cellularity in the latter. These results suggest that combined treatment with bioimplants and BMNCs have an additive effect on massive bone defects in lambs. These experimental results could be applied to repair damaged human bone.
1. Introduction
shape, and size. Additionally, the patient undergoes a second surgical procedure, thus increasing morbidity by 10% (Younger and Chapman, 1989). The use of allografts has increased in the last 20 years, but these are also far from ideal because of the lack of complete integration, especially for large defects (Bus et al., 2014); this problem leads to its main complications, such as fractures, infections, and resorptions (Ayvaz et al., 2014; Delloye et al., 2014; San-Julian and Canadell, 1998). The recent development of “tissue engineering” offers new options for repairing bone defects (Langer and Vacanti, 1993) and one of the corresponding fields is the creation of three-dimensional bioimplants that provide a suitable environmental medium for tissue growth (Cunniffe et al., 2010; Hansen et al., 2005). Demineralised bone matrix
Since the beginning of the 1970s, when the era of limb salvage began (Eilber et al., 1980), different forms of reconstruction have been used to treat massive bone defects, ranging from the use of an autograft, allografts (Aponte-Tinao et al., 2014, 2015, 2016; Houdek et al., 2016; Rabitsch et al., 2013), bone substitutes, special prostheses (Gkavardina and Tsagozis, 2014; Tiwari et al., 2014), or combinations of these reconstruction methods (Antoci et al., 2009; Arslan et al., 2015; Qu et al., 2015; Sevelda et al., 2015; Venkatramani et al., 2015; Vlad et al., 2013). Autologous bone remains the gold standard for bone grafts; however, it has the enormous disadvantage of being limited in quantity,
⁎ Corresponding author at: Servicio de Traumatología y Ortopedia, Hospital Universitario "Dr. José Eleuterio González", Universidad Autónoma de Nuevo León, Av. Madero y Gonzalitos S/N Colonia Mitras Centro, Monterrey, N.L., México. A. P.1563. E-mail address: [email protected] (E. Álvarez-Lozano).
https://doi.org/10.1016/j.tice.2017.12.005 Received 23 September 2017; Received in revised form 7 December 2017; Accepted 9 December 2017 Available online 12 December 2017 0040-8166/ © 2017 Elsevier Ltd. All rights reserved.
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[DBM] (Urist, 1965; Vaccaro et al., 2002) is a very useful material because it has bone morphogenetic proteins [BMPs] (Urist et al., 1970; Urist and States, 1971), which act as osteoinductive mediators; however, an osteoinductive agent without a carrier will be lost or degraded rapidly without producing any osteogenic effect (Duflo et al., 2006; Hotz and Herr, 1994; Liu et al., 2006). Because bone is a nanostructured tissue (Balasundaram and Webster, 2007), this carrier must have certain chemical and structural characteristics (Seref-Ferlengez et al., 2015; Song et al., 2015; Terauchi et al., 2015). “Stem cells” (Nowakowski et al., 2015) have recently aroused a growing interest in their therapeutic potential in diseases that until now lacked an effective treatment. These cells are found in the quiescent state in the body, mainly in bone marrow and adipose tissue, as multipotent stromal cells (Maumus et al., 2011; O'Brien et al., 2007). The structure, cellularity, function, and biological mechanisms of bone tissue make it difficult to find a material, biological or otherwise, that replaces bone and replicates all its characteristics. It is important to provide a biological and structural aid to the body to facilitate the repair of massive bone defects through fast, high-quality integration. The aim of this study was to evaluate if a three-dimensional bioimplant improves the speed and quality of integration in the recipient bones, compared to allograft treatment, of massive bone defects. This bioimplant was generated from the combination of the components of bone tissue: DBM with BMPs, collagen, hydroxyapatite [HAp] Ca10(PO4)6(OH)2, and osteoprogenitor cells.
proximal diaphysis of the left tibia of the lambs using an oscillating saw and a metallic guide specifically fabricated for this purpose. The bone defect comprised 75% of the cortical circumference and 5 cm of the length. The bone defects were treated in three different manners; animals were randomly selected and divided into 3 groups of five lambs each. Group one lambs were treated only with the bioimplant. Group two lambs were treated with the bioimplant plus autologous nucleated cells; prior to its placement, the bioimplant was immersed for 5 min in a container with the cells obtained from an iliac crest aspirate by a special centrifugation process a few hours before surgery. In group three, the defect was treated with a frozen allograft. In all cases, internal fixation with a special plate and screws was used to keep the allograft in place. The lambs remained in the bioterium for 5 days for wound monitoring and administration of antibiotic (20 mIU procaine benzylpenicillin) without restriction of limb support and with ad libitum access to water and food. From the 6th day onwards, the animals were transferred to a common habitat. The experiments were performed in accordance with the International Guidelines on the Appropriate Use of Experimental Animals and according to the Mexican Norm NOM-062-ZOO-1999 on the Technical Specifications for the Production, Care and Use of Laboratory Animals (SAGARPA, 1999). The protocol was approved by the Bioethical Committee of the Faculty of Medicine, UANL in Monterrey, Nuevo León, México.
2. Materials and methods
2.4. Radiological analysis
2.1. Study design and setting This is an experimental, longitudinal, comparative, blind and controlled study.
In the radiological analysis, antero-posterior and lateral radiographs of the limb were taken at weeks 1 (control), 6, and 12 post-treatment and the appearance of the massive bone defects in the tibias of the study groups were evaluated.
2.2. Preparation of bioimplant
2.5. Histological and histochemical analysis
The first component used for the bioimplant was DBM, which was generated via the demineralisation of the diaphysis of the long bones of 3 hind limbs of donor lambs using a technique previously described by Rivera et al. (2003). The DBM has biological characteristics that favour osteoinduction (Urist et al., 1970; Urist and Strates, 1971; Vaccaro et al., 2002). The second component was collagen because collagen is the most abundant structural protein in bone; collagen was used to provide rigidity and maintain the desired shape of the bioimplant while making the implant insoluble when contacting body fluids. Collagen was obtained from the skin of foetal pigs by a process consisting of mechanically macerating the skin in a meat grinder and dripping a 0.5% by weight solution of hydrochloric acid [HCl] with constant stirring in distilled water. Bone is a nanostructured tissue, and therefore to achieve a porosity of less than 100 nm, HAp nanoparticles were selected as the third component. HAp is the main inorganic component of bone and its use in nanoparticles improves the microenvironment and increases the useful surface area for cell growth. The HAp nanoparticles were processed from waste egg shells by a hydrothermal synthesis process, as described in the technique published by Elizondo-Villarreal et al. (2012). The three components were mixed in a proportion of 30% DBM, 60% collagen, and 10% HAp. The mixture was placed in prefabricated 5-cc moulds of a specific shape and size, dried, and subsequently sterilised in doses of 20–22 kg.
Animals euthanized with sodium pentobarbital (90–210 mg/kg weight, intravenous administration) at 12 weeks to obtain the studied bone segment for morphological analyses. After the samples were collected, they were fixed in 4% paraformaldehyde solution in 1X phosphate buffered saline [PBS] pH 7.2–7.4 for 24 h. Subsequently, a 1-cmthick segment was collected and treated with the 10% HCl decalcification technique for 21 days by changing the solution every third day, observing that the bone showed a soft consistency. Afterward, samples were processed by conventional histological techniques until their inclusion in paraffin blocks. The general cellular characteristics, presence of bone trabeculae, and orientation and organisation of the collagen fibres were evaluated in histological sections (4 μm thick) stained with haematoxylin and eosin [H&E] and Mallory-Azán Trichrome [M-AT] for histological analysis. Additionally, the histochemical periodic acid-Schiff [PAS] staining method was used to identify the components of the osteoid. Samples were evaluated using light field microscopy. 2.6. Immunohistochemical analysis To identify the presence of osteoblasts in the samples of interest, immunohistochemical analysis was performed in 4-μm-thick histological sections immunolabeled with polyclonal anti-Parathyroid Hormone Receptor R1[PTHR/PTHR1] antibody (aa388-406, LSC313515 (1:200), LifeSpan Biosciences, Inc., Seattle, WA, USA). An Abcam® anti-mouse & rabbit HRP/DAB detection kit (ab64264 Cambridge, MA, USA) was used as the detection system. Positivity was visualised with 3,3′-diaminobenzidine [DAB] and the nuclei were identified using Gill’s haematoxylin.
2.3. Description of experiment, treatment, or surgery We employed 15 female lambs (Ovis aries), 4–5 months of age and weighing 20–30 kg. A bone defect was surgically created in the 70
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weeks 1 (control), 6, and 12 post-treatment for radiological evaluation. The radiographs were evaluated by 3 different orthopaedic doctors and they gave 1 point if the x-ray showed no ossification or integration, 2 points if the ossification or integration was less than 50%, and 3 points if they found more than 50% ossification or integration. The anteroposterior radiograph in the first postoperative week with a bioimplant plus the nucleated cells of autologous bone marrow showed the bone defect (Fig. 1E). At week 12, the same limbs showed 100% ossification (Fig. 1F). We didńt find any statistical difference between the groups.
2.7. Morphometric analysis To quantify the osteoid percentage, the samples were grouped according to the type of treatment without specifying the treatment; 40 consecutive fields were photodocumented in each group with a strong dry objective (40×) in the PAS-positive samples (8 fields/slice, 1 slice/ lamb, and 5 lambs/group). High-resolution digital images were obtained with a Nikon Eclipse 50i microscope and a Digital Sight dDS2Mu image analysis system with NIS-Elements software. Subsequently, the images were analysed with ImageJ version 1.49 (National Institutes of Health); colour, tone distribution, saturation, and illumination were the same for all the images. This morphometric analysis allowed us to determine the osteoid percentage in the study groups. Subsequently, in these same images, the intensity [IntDent]of the positivity in the osteoid in each sample was quantified by microdensitometry. The values obtained were expressed as optical density. The mean value and the standard deviation [SD] of all the values from the morphometric analyses were obtained for statistical analysis and for comparison between the study groups. In addition, morphometric analysis of PTHR/PTHR1-positive cells was performed in which the positive cells/field were quantified in 40 fields for each group with the same methodology described previously. The morphological analysis of the samples was performed by two specialists in morphology who were blinded to the treatment used in each sample and blinded to each other.
3.3. Histological analysis In the histological analysis of the group 1 implantation site (bioimplant), we observed areas with different collagen fibre organisation and cell layout, so they were classified into three areas according to the cellular and tissue organisation (Fig. 2A). Zone 1 showed irregularly organised fibres with cells between and at the periphery of the bundles; notably, in this zone, the spaces are similar to small blood vessels between the collagen fibres (Fig. 2D). In zone 2, a larger organisation of the fibres and cells was observed, as well as larger-diameter spaces (Fig. 2G). Finally, in zone 3, the histological characteristics of an immature bone tissue are clearly observed: bundles of collagen fibres, which began to organise around a cavity with cells in its interior, similar to the endosteum of the Haversian canals. In addition, round cells similar to osteocytes were observed in the gaps between the fibres (Fig. 2J). An important finding in this group was the presence of areas of acidophilic material that represent osteoid (Fig. 2D). In group 2 (bioimplant+ bone marrow nucleated cells), the same previously-described areas were observed (Fig. 2B). Notably, a higher cellularity was observed in this group compared to group 1 (Fig. 2E). Moreover, in this group, the organisation of osteon-like structures was greater than in group 1 (Fig. 2H and K) and a greater amount of osteoid was observed (Fig. 2E). Finally, in group 3 (allograft), clear divisions were observed between the bone tissue laminae with a distinct organisation of the osteons (Fig. 2C). In this group, endosteal remnants were observed in the Haversian canals (Fig. 2F and I) and there were no cells in the gaps of the concentric laminae (Fig. 2L). The previously-described results were corroborated with the M-AT stain. Pale red lines indicative of an osteoid were observed in the
3. Results 3.1. Characterisation of the implant Prior to implantation, the bioimplant was characterised; we observed collagen fibre bundles with acidophilic staining and cells with basophilic nuclei (Fig. 1A and B). These data were confirmed by the MAT stain showing the blue collagen fibres and cells with red cytoplasm (Fig. 1C). Also, in the bioimplant samples, we observed small, semitransparent granular structures corresponding to the DBM and HAp (Fig. 1D). 3.2. Radiological analysis Antero-posterior and lateral radiographs of the limbs were taken at
Fig. 1. Morphological characterisation of bioimplant: A and C) Panoramic images showing bundles of collagen fibres. B and D) Amplification of the boxes showing the collagen fibres (red arrows) and demineralised bone matrix (yellow arrow). Light microscopy. Inclusion in paraffin. A and B: H&E stain; C and D: M-AT stain. E and F): Antero-posterior X-ray of lamb tibia, 1 week (E) and 12 weeks (F) after bone defect treated with bioimplant plus nucleated cells from bone marrow, showing 100% ossification. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. The use of bioimplant has an osteogenic effect in vivo: A, B and C) Panoramic images of group 1, group 2, and group 3. D and G) Amplifications of boxes 1 and 2 of image A, collagen fibre bundles (blue arrows) with cells between the bundles (yellow arrows) and at the periphery (white arrows), and small vessels (green arrows). J) Amplification of box 3 of image A corresponding to zone 3, which shows osteons with concentric laminae (blue arrow) around blood vessels (green arrow) and with osteocytes in their lagoons (yellow arrow). E and H) Amplifications of boxes 1 and 2 of image B, with the same elements indicated for group 1. K) Amplification of box 3 of image B corresponding to zone 3.There is a greater organisation of osteons with concentric sheets (blue arrow) around areas resembling small blood vessels (green arrow) and with osteocytes in their lagoons (yellow arrow) than in group 1. F, I and L) Amplifications of boxes 1, 2, and 3 of image C, plaques of bone tissue (blue arrows), with spaces without cells (white arrows) and endosteal remnants in the Havers canals (green arrows). Light microscopy. Inclusion in paraffin. H&E stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Histochemical analysis
periphery of the developing bone trabeculae or in areas that contained cells in groups 1 and 2 with this staining (Fig. 3A, D, G and J); this feature was more pronounced in group 2 (Fig. 3B, E, H and K). On the other hand, in group 3, few areas with the previously described characteristics of an osteoid were observed via this staining (Fig. 3C, F, I and L).
In addition, this method permitted the identification of positivity in the cells of both group 1 (Fig. 4 and D) and group 2 (Fig. 4B and E). It should be noted that this stain allowed the identification of extensive areas with positivity in developing bone trabeculae as well as areas with amorphous PAS material suggestive of extracellular matrix 72
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Fig. 3. Bioimplant induces osteoid secretion: A, B and C) Panoramic images of study groups. D) Amplification of box 1 of image A, collagen fibre bundles (green arrow) with cells (yellow arrow). G and J) In zones 2 and 3, there is a greater organisation of fibres similar to osteons with concentric laminae (green arrows), cells (yellow arrows), and pale red lines indicative of an osteoid in the periphery of developing bone trabeculae or in areas that contained cells in red (red arrows). E and H) Amplifications of boxes 1, 2 and 3 of image B, with the same elements indicated for group 1. K) There is a greater organisation of osteons with concentric laminae (green arrow), there are osteocytes in their lagoons (yellow arrow), and osteoid staining was more pronounced (red arrow). F, I and L) Amplifications of boxes 1, 2 and 3 of image C, plaques of bone tissue (green arrow), with spaces without cells (yellow arrows) and few areas with osteoid characteristics (red arrow). Light microscopy. Inclusion in paraffin.M-AT stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
program, the following values were observed: 4.429E + 07 ± 3.135E + 07 for group 1, 1.554E + 08 ± 4.07E + 07 for group 2, and 4.14E + 06 ± 2.15E + 06 for group 3. As shown in the graph, group 2 showed the highest intensity, followed by group 1 and finally group 3 (Fig. 5B).
synthesised by the cellular elements (Fig. 4G, J, H, and K). Interestingly, greater positivity was observed in group 2 than in groups 1 and 3, with the latter showing few areas of positivity (Figs. 4C, F, I, and L). In the morphometric analysis of PAS histochemical positivity in the osteoid of each of the study groups, values of 5.831% ± 2.240% were observed in group 1, 16.585% ± 3.597 in group 2, and 1.316% ± 0.865 in group 3 (Fig. 5A). When the intensity of the density was quantified using the ImageJ 73
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Fig. 4. The use of bioimplant activates osteoid synthesis in vivo: A, B and C) Panoramic images of groups. D, G and J) Amplifications of zones 1, 2 and 3 of image A, cells with positivity in the cytoplasm (green arrows) and positive material between fibres and trabeculae (blue arrow). E, H and K) Amplifications of boxes 1, 2 and 3 of image B, with the same elements identified for group 1. Greater positivity was observed in group 2. F, I and L) Amplifications of the boxes 1, 2 and 3 of image C, thin lines of osteoid (blue arrows). Light microscopy. Inclusion in paraffin. PAS stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In the morphometric analysis of the positive cells, values of 8.175 ± 0.734 were observed for group 1, 11.475 ± 0.531 for group 2, and 1.175 ± 0.090 for group 3. These results demonstrate that the number of osteoblasts was highest in group 2 (Fig. 6G).
3.5. Immunohistochemical analysis The immunolabeling with anti-PTHR/PTHR1-specific antibodies showed areas with positive cells, indicating the presence of osteoblasts; these cells were observed mainly in the samples of groups 1 and 2. Positivity was observed in cells situated around the trabeculae, between the collagen fibres, and in the interior of the spaces, suggestive of vessels or Haversian canals (Fig. 6A–D). Group 3 showed few positive cells (Fig. 6E and F). The bioimplant showed negative results for osteoblast cells (data not shown).
3.6. Statistical analysis, study size The Kolmogorov-Smirnov test showed that the samples were not normally distributed; therefore, we used the Kruskal-Wallis test to compare the groups. A post-hoc test was used and we found a statistical 74
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Fig. 5. Bioimplant shows an increased quantity of osteoid synthesis: A) Morphometric analysis of the osteoid of each of the study groups. Group 2 shows a higher percentage of osteoid than the other groups. B). Morphometric analysis of density of osteoid. Group 2 showed the highest density, followed by group 1 and finally group 3. Statistical differences were found betweenthe groups. * p ≤ 0.05.
may be occurring in this model’s process of regeneration that results in the formation of bone tissue. On the other hand, a greater amount of concentric laminae with spaces similar to Havers' canals characteristic of the osteons of bone tissue was observed in group 2, compared to groups 1 and 3. These results suggest that the use of HAp promotes the organisation of the fibres and cells. Studies with materials for bone repair using an aggregate of mineral trioxide combined with monoolein gel demonstrate the synthesis of bone tissue in defects caused in the jaws of experimental animals (Falcão-Filho et al., 2007). The samples analysed with the M-AT stain had the histological characteristics of immature bone tissue. These results agree with those described for bone-remodelling units [BRUs], including osteoblasts. In the areas with BRUs activity, the osteoid synthesis is observed as red lines on the trabeculae, which were identified with trichrome methods (Velásquez-Forero, 2009). The cells of groups 1 and 2 were PAS-positive in the cytoplasm and there were areas of PAS-positive material with and without the presence of cells. These results suggest that the cells present in the area of the bioimplant synthesise and secrete components of the extracellular matrix. In addition, DBM proteins have been reported to act as modulators of growth factors (Young, 2003). Because the matrix proteins
significance between all groups, group 1 vs group 2, group 1 vs group 3, and group 2 vs group 3.
4. Discussion The results of the present study demonstrate for the first time that implantation of a biomaterial that does not have a specific structure, similar to our bioimplant, in a bone repair model has an osteogenic effect; promoting the synthesis of extracellular matrix and the organisation of collagen fibres in concentric layers around vascular spaces, similar to the histological characteristics of bone tissue. These characteristics were observed after only 3 months of bioimplantation in the in vivo model. In this study, samples of groups 1 and 2 showed large areas with eosinophilic material corresponding to osteoid, with and without the presence of cells around and between the trabeculae of the bone tissue in formation. These findings are consistent with those observed in studies that describe osteogenic events during embryogenesis and later in bone regeneration: cellular aggregates that subsequently differentiate from precursor cells into an osteoblastic lineage, which then deposit and mineralise the osteoid and finally differentiate from osteoblasts to osteocytes (Kaul et al., 2015). Therefore, these phenomena 75
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Fig. 6. Bioimplant shows an increased number of osteoblasts in vivo: A-F) Immunolabeling with anti-PTHR/PTHR1-specific antibodies in groups 1, 2 and 3.Areas with positive cells indicating the presence of osteoblasts (yellow arrows) and negative cells (green arrows). These cells were mainly observed in the samples of groups 1 (A and B) and 2 (C and D). Group 3 showed few positive cells (E and F). Light microscopy. Inclusion in paraffin. Immunohistochemistry. G) Quantification of positive cells/field in each study group. An increased number of positive cells were observed in group 2. Statistical differences were found between the groups. * p ≤ 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
bioimplant. The morphogenic proteins are abundant in bone tissue, and we know that BMPs are involved in the formation of bone and cartilage during embryogenesis (Fernandez-Tresguerres-Hernandez-Gil et al., 2006). Also, BMPs are now considered one the most potent factors of osteoblastic differentiation (Yamaguchi et al., 2000). Canalis et al. reported that in addition to stimulating osteogenesis, morphogenic proteins inhibit osteoclasto genesis (Canalis et al., 2003). Furthermore, the induction of bone regeneration has been described in a model of mandibular distraction surgery treated with transduced mesenchymal stem cells with the human morphogenic protein-2
are at a concentration 1000 times greater than the growth factors, these proteins could play a more important role in the regulation of different cellular functions (Horowitz, 2003). We did not find reports in the literature that described these phenomena that are induced by the components of the bioimplant used in this study. Finally, some cells were identified by immunolabeling with antiPTHR1-specific antibodies as osteoblasts. These results suggest that the use of our bioimplant induces the differentiation and/or migration of cells. In this study we used the DBM of lamb with BMPs as part of the
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(Castro-Govea et al., 2012). Moreover, BMPs also induce the differentiation of osteoprogenitor cells and participate in the regulation of osteoblastic cell proliferation, migration, and differentiation into osteocytes (Fernandez-TresguerresHernandez-Gil et al., 2006). To date, the mechanisms of cell differentiation and stimulation of osteoid synthesis observed in this study remain unknown; however, these results will be useful for the development of new therapeutic strategies that seek to shorten the time of bone repair and to obtain a greater organisation of the cellular and fibrous elements present in the bone tissue.
Balasundaram, G., Webster, T.J., 2007. An overview of nano-polymers for orthopedic applications. Macromol. Biosci. 7, 635–642. Bus, M.P., Dijkstra, P.D., van de Sande, M.A., Taminiau, A.H., Schreuder, H.W., Jutte, P.C., van der Geest, I.C., Schaap, G.R., Bramer, J.A., 2014. Intercalary allograft reconstructions following resection of primary bone tumors: a nationwide multicenter study. J. Bone Joint Surg. Am. 96, e26. Canalis, E., Economides, A.N., Gazzerro, E., 2003. Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr. Rev. 24, 218–235. Castro-Govea, Y., Cervantes-Kardasch, V.H., Borrego-Soto, G., Martínez-Rodríguez, H.G., Espinoza-Juárez, M., Romero-Díaz, V., Marino-Martínez, I.A., Robles-Zamora, A., Álvarez-Lozano, E., Padilla-Rivas, G.R., 2012. Human bone morphogenetic protein 2–transduced mesenchymal stem cells improve bone regeneration in a model of mandible distraction surgery. J. Craniofac. Surg. 23, 392–396. Cunniffe, G.M., Dickson, G.R., Partap, S., Stanton, K.T., O’Brien, F.J., 2010. Development and characterization of a collagen nano-hydroxyapatite composite scaffold for bone tissue engineering. J. Mater. Sci. Mater. Med. 21, 2293–2298. Delloye, C., van Cauter, M., Dufrane, D., Francq, B.G., Docquier, P.L., Cornu, O., 2014. Local complications of massive bone allografts: an appraisal of their prevalence in 128 patients. Acta Orthop. Belg. 80, 196–204. Duflo, S., Thibeault, S.L., Li, W., Shu, X.Z., Prestwich, G.D., 2006. Vocal fold tissue repair in vivo using a synthetic extracellular matrix. Tissue Eng. 12, 2171–2180. Eilber, F.R., Mirra, J.J., Grant, T.T., Weisenburger, T., Morton, D.L., 1980. Is amputation necessary for sarcomas? A seven-year experience with limb salvage. Ann. Surg. 192, 431–438. Elizondo-Villarreal, N., Martínez-de-la-Cruz, A., Guerra, R.O., Gómez-Ortega, J.L., TorresMartínez, L.M., Castaño, V.M., 2012. Biomaterials from agricultural waste: eggshellbased hydroxyapatite. Water Air Soil Pollut. 223, 3643–3646. Falcão-Filho, H.B.L., Issa, J.P.M., Nascimento, C., Iyomasa, M.M., Regalo, S.C.H., Siéssere, S., Pedrazzi, V., Pitol, D.L., 2007. Histological evaluation of the bone repair using mineral trioxide aggregate combined to a material carrier. Int. J. Morphol. 25 (4), 789–796. Fernandez-Tresguerres-Hernandez-Gil, I., Alobera-Gracia, M.A., del-Canto-Pingarron, M., Blanco-Jerez, L., 2006. Physiological bases of bone regeneration II. The remodeling process. Med. Oral Patol. Oral Cir. Bucal 11, E151–E157. Gkavardina, A., Tsagozis, P., 2014. The use of megaprostheses for reconstruction of large skeletal defects in the extremities: a critical review. Open Orthop. J. 8, 384–389. Hansen, J.K., Thibeault, S.L., Walsh, J.F., Shu, X.Z., Prestwich, G.D., 2005. In vivo engineering of the vocal fold extracellular matrix with injectable hyaluronic acid hydrogels: early effects on tissue repair and biomechanics in a rabbit model. Ann. Otol. Rhinol. Laryngol. 114, 662–670. Horowitz, M., 2003. Matrix proteins versus cytokines in the regulation of osteoblasts function and bone formation. Calcif. Tissue Int. 72, 5–7. Hotz, G., Herr, G., 1994. Bone substitute with osteoinductive biomaterials–current and future clinical applications. Int. J. Oral Maxillofac. Surg. 23, 413–417. Houdek, M.T., Wagner, E.R., Stans, A.A., Shin, A.Y., Bishop, A.T., Sim, F.H., Moran, S.L., 2016. What is the outcome of allograft and intramedullary free fibula (Capanna technique) in pediatric and adolescent patients with bone tumors? Clin. Orthop. Relat. Res. 474, 660–668. Kaul, H., Hall, B.K., Newby, C., Ventikos, Y., 2015. Synergistic activity of polarised osteoblasts inside condensations cause their differentiation. Sci. Rep. 5, 11838. Langer, R., Vacanti, J.P., 1993. Tissue engineering. Science 260, 920–926. Liu, Y., Ahmad, S., Shu, X.Z., Sanders, R.K., Kopesec, S.A., Prestwich, G.D., 2006. Accelerated repair of cortical bone defects using a synthetic extracellular matrix to deliver human demineralized bone matrix. J. Orthop. Res. 24, 1454–1462. Maumus, M., Peyrafitte, J.A., D'Angelo, R., Fournier-Wirth, C., Bouloumie, A., Casteilla, L., Sengenes, C., Bourin, P., 2011. Native human adipose stromal cells: localization, morphology and phenotype. Int. J. Obes. 35, 1141–1153. Nowakowski, A., Walczak, P., Janowski, M., Lukomska, B., 2015. Genetic engineering of mesenchymal stem cells for regenerative medicine. Stem Cells Dev. 24, 2219–2242. O'Brien, F.J., Harley, B.A., Waller, M.A., Yannas, I.V., Gibson, L.J., Prendergast, P.J., 2007. The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering. Technol. Health Care 15, 3–17. Qu, H., Guo, W., Yang, R., Tang, X., Yan, T., Li, D., Yang, Y., Zang, J., 2015. Cortical strut bone grafting and long-stem endoprosthetic reconstruction following massive bone tumour resection in the lower limb. Bone Joint J. 97B, 544–549. Rabitsch, K., Maurer-Ertl, W., Pirker-Frühauf, U., Wibmer, C., Leithner, A., 2013. Intercalary reconstructions with vascularised fibula and allograft after tumour resection in the lower limb. Sarcoma 2013, 8. Rivera, J.A., Benavides, C.R., Monsalve, P.A., Injertos, Osorio A., 2003. óseos-Nueva alternativa: Fase I. Extracción de proteínas morfogenéticas parcialmente purificadas de hueso bovino. Rev. Colomb. Cienc. Pec. 16, 139–146. San-Julian, M., Canadell, J., 1998. Fractures of allografts used in limb preserving operations. Int. Orthop. 22, 32–36. Seref-Ferlengez, Z., Kennedy, O.D., Schaffler, M.B., 2015. Bone microdamage, remodeling and bone fragility: how much damage is too much damage? Bonekey Rep. 4, 644. Sevelda, F., Schuh, R., Hofstaetter, J.G., Schinhan, M., Windhager, R., Funovics, P.T., 2015. Total femur replacement after tumor resection: limb salvage usually achieved but complications and failures are common. Clin. Orthop. Relat. Res. 473, 2079–2087. Song, W., Li, X., Qian, J., Lv, G., Yan, Y., Su, J., Wei, J., 2015. Mesoporous calcium-silicon xerogels with mesopore size and pore volume influence hMSC behaviors by load and sustained release of rhBMP-2. Int. J. Nanomed. 10, 1715–1726. Terauchi, M., Ikeda, G., Nishida, K., Tamura, A., Yamaguchi, S., Harada, K., Yui, N., 2015. Supramolecular polyelectrolyte complexes of bone morphogenetic protein-2 with sulfonated polyrotaxanes to induce enhanced osteogenic differentiation. Macromol. Biosci. 15, 953–964.
5. Conclusions The results observed in this study allow us to propose the use of our bioimplant with autologous nucleated bone marrow cells for bone repair because this combination showed a stronger osteogenic effect in the in vivo model, as analysed by morphological and morphometric tests, than did the cell-free bioimplant or the use of an allograft. 6. Background and rationale At the present time there is no material, biological tissue or otherwise, with the structure, cellularity, function, and biological mechanisms of bone tissue. We are not trying to find “the magic tissue” that replaces bone and replicates all its characteristics. Our goal is to provide the body with a biological and structural aid to facilitate the repair of massive bone defects while producing higher-quality repairs that require less time to heal. 7. Limitations The main limitation of this study was that we did not use flow cytometry to analyse cells obtained from an iliac crest aspirate and name them “stem cells”; instead, we named them autologous nucleated cells from iliac crest, even knowing that many of these cells are “stem cells”. Conflicts of interest None. Acknowledgements We thank Federico A. Rodríguez Ph.D. and Nora Elizondo Ph.D. for their investigation in biomaterials and donation of nanoparticles, and Jorge Lozano for his contribution regarding the investigation animals.We also thank Luis I. Botello S. and Iván A. Mesta R. for technical assistance involved in processing the samples for morphological analysis. References Antoci, V., Phillips, M.J., Antoci Jr., V., Krackow, K.A., 2009. Using an antibiotic-impregnated cement rod-spacer in the treatment of infected total knee arthroplasty. Am. J. Orthop. 38, 31–33. Aponte-Tinao, L.A., Ritacco, L.E., Albergo, J.I., Ayerza, M.A., Muscolo, D.L., Farfalli, G.L., 2014. The principles and applications of fresh frozen allografts to bone and joint reconstruction. Orthop. Clin. North Am. 45, 257–269. Aponte-Tinao, L., Ayerza, M.A., Muscolo, D.L., Farfalli, G.L., 2015. Survival, recurrence, and function after epiphyseal preservation and allograft reconstruction in osteosarcoma of the knee. Clin. Orthop. Relat. Res. 473, 1789–1796. Aponte-Tinao, L.A., Ayerza, M.A., Muscolo, D.L., Farfalli, G.L., 2016. What are the risk factors and management options for infection after reconstruction with massive bone allografts? Clin. Orthop. Relat. Res. 474, 669–673. Arslan, H., Ozkul, E., Gem, M., Alemdar, C., Sahin, I., Kisin, B., 2015. Segmental bone loss in pediatric lower extremity fractures: indications and results of bone transport. J. Pediatr. Orthop. 35, e8–e12. Ayvaz, M., Bekmez, S., Mermerkaya, M.U., Caglar, O., Acaroglu, E., Tokgozoglu, A.M., 2014. Long-term results of reconstruction with pelvic allografts after wide resection of pelvic sarcomas. Sci. World J. 2014, 6.
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C.E. Cuervo-Lozano et al.
Venkatramani, H., Sabapathy, S.R., Dheenadayalan, J., Devendra, A., Rajasekaran, S., 2015. Reconstruction of post-traumatic long segment bone defects of the lower end of the femur by free vascularized fibula combined with allograft (modified Capanna's technique). Eur. J. Trauma Emerg. Surg. 41, 17–24. Vlad, C., Gavriliu, T., Georgescu, I., Dan, D., Parvan, A., Burnei, G., 2013. Bone transport with the lengthening through the physis in patients having congenital pseudarthrosis of tibia – short-term results. J. Med. Life. 6, 266–271. Yamaguchi, A., Komori, T., Suda, T., 2000. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr. Rev. 21, 393–411. Young, M.F., 2003. Bone matrix proteins: more than markers. Calcif. Tissue Int. 72, 2–4. Younger, E.M., Chapman, M.W., 1989. Morbidity at bone graft donor sites. J. Orthop. Trauma 3, 192–195.
Tiwari, A., Jain, S., Mehta, S., Kumar, R., Kapoor, G., Kumar, K., 2014. Limb salvage surgery for osteosarcoma: early results in Indian patients. Indian J. Orthop. 48, 266–272. Urist, M.R., Strates, B.S., 1971. Bone morphogenetic protein. J. Dent. Res. 50, 1392–1406. Urist, M.R., Jurist Jr., J.M., Dubuc, F.L., Strates, B.S., 1970. Quantitation of new bone formation in intramuscular implants of bone matrix in rabbits. Clin. Orthop. Relat. Res. 68, 279–293. Urist, M.R., 1965. Bone: formation by autoinduction. Science 150, 893–899. Vaccaro, A.R., Chiba, K., Heller, J.G., Patel, T., Thalgott, J.S., Truumees, E., Fischgrund, J.S., Craig, M.R., Berta, S.C., Wang, J.C., 2002. Bone grafting alternatives in spinal surgery. Spine J. 2, 206–215. Velásquez-Forero, F.H., 2009. Histomorfometría de la biopsia ósea previo marcaje y procesada sin descalcificar. Patología 47, 108–117.
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