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Osteostatin-loaded onto mesoporous ceramics improves the early phase of bone regeneration in a rabbit osteopenia model
Acta Biomaterialia 8 (2012) 2317–2323
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Osteostatin-loaded onto mesoporous ceramics improves the early phase of bone regeneration in a rabbit osteopenia model Daniel Lozano a,b,1, Cynthia G. Trejo c, Enrique Gómez-Barrena d,1, Miguel Manzano e,f, Juan C. Doadrio e,f, Antonio J. Salinas e,f, María Vallet-Regí e,f,⇑, Natalio García-Honduvilla c, Pedro Esbrit a,b, Julia Buján c a
Laboratorio de Metabolismo Mineral y Óseo, Instituto de Investigación Sanitaria (IIS)-Fundación Jiménez Díaz, 28040 Madrid, Spain Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF), Instituto de Salud Carlos III, 28029 Madrid, Spain Departamento de Especialidades Médicas, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain d Departamento de Cirugía Ortopédica y Traumatología, Hospital La Paz, Universidad Autónoma de Madrid, 28046 Madrid, Spain e Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain f Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain b c
a r t i c l e
i n f o
Article history: Received 16 November 2011 Received in revised form 1 March 2012 Accepted 6 March 2012 Available online 10 March 2012 Keywords: Osteoporosis Osteostatin Bone regeneration SBA15 mesoporous silica
a b s t r a c t Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Recently, we reported that PTHrP (107–111) (osteostatin) coating onto mesoporous ceramics confers osteogenic activity to these materials. Bone repair is dramatically compromised in osteopenia/osteoporosis. Thus, we examined the efficacy of unmodified and organically modified SBA15 ceramics loaded with osteostatin in promoting bone repair in an osteoporotic rabbit model. Osteoporosis was induced in New Zealand rabbits by methylprednisolone administration, and healthy rabbits were used as controls. Tested materials were implanted into a femoral cavitary defect, and animals were sacrificed at 2 weeks post-implantation. At this time, implants were encapsulated by a variable layer of fibrotic tissue with no evidence of inflammation. Similarly to observations in normal rabbits, both types of osteostatin-loaded bioceramics induced tissue regeneration associated with increased staining for PCNA, Runx2, osteopontin, and/or vascular endothelial growth factor in osteoporotic rabbits. Our present findings demonstrate that these osteostatinbearing bioceramics increase the early repair response not only in normal bone but also in osteoporotic bone after a local injury. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Bone repair to reconstitute skeletal integrity begins immediately after trauma. The healing events involve a hematoma-related infiltration of inflammatory cells, which increases the local delivery of cytokines at the site of damage, promoting both the recruitment and growth of mesenchymal stem cells from the periostial cambium [1,2]. These cells then respond by committing to the chondrogenic or osteogenic lineage, depending on whether vasculature is disrupted (close to the bone disjunction) or better preserved, respectively; the latter process is also favoured by the mechanical stability of the injured bone. Thus, the repair process is classically thought to involve both intramembranous and endochondral ossification followed by osteoclast-directed
⇑ Corresponding author at: Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. Tel.: +34 91 394 1861; fax: +34 91 394 1786. E-mail address: [email protected] (M. Vallet-Regí). 1 Present address: Grupo de Investigación de Cirugía OsteoArticular, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), 28046 Madrid, Spain.
remodelling [3]. Recent evidence also supports the notion that at an early stage during bone regeneration, recruitment of stromal cells may activate osteoclastogenesis and osteoclast-mediated resorption of damaged bone. This sets the conditions for new bone formation in a way that mimics bone remodelling within the basic multicellular units in the bone surface [4,5]. Knowledge of the molecular pathways and factors implicated in bone regeneration after skeletal trauma is still limited. However, the important roles played by bone morphogenetic proteins (BMPs), namely BMP-2 [2,6], and, as shown more recently, by Wnt/b-catenin pathway factors [7], in this regard provide a molecular rationale for using these factors as osteoinductive therapies. Injured bone tissue revascularization is a source of oxygen, nutrients and cell precursors, and thus is critical for successful bone repair. This is mainly carried out by the production of vascular endothelial growth factor (VEGF) in response to BMPs and other local factors in the callus, establishing the basis for VEGF administration as a putative therapy to enhance skeletal healing [8,9]. Other factors such as interleukins-1 and -6 and receptor activator of NF-jB ligand have an important role in osteoclast recruitment and newly formed bone remodelling [5,10].
1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.03.014
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Much less is currently known about the true underlying mechanisms of the observed reduction of bone repair in the setting of osteopenia. In this regard, an impaired fracture healing occurs with aging, which might be caused at least in part by a decrease in both osteogenic commitment of mesenchymal cells and angiogenesis [11,12]. Moreover, bone regeneration and bone repair have been shown to be dramatically hampered in diabetic conditions, associated with a decreased osteoblastic function [13,14]. This deficit can be reversed by systemic administration of insulin or parathyroid hormone (PTH)-related protein (PTHrP), an important modulator of bone formation and bone remodelling [14,15]. Moreover, this type of administration of PTH or PTHrP was shown to promote bone repair in ovariectomized rats [16–18] or rabbits with glucocorticoid-related osteopenia [19], respectively. Furthermore, it was recently shown that every other day injection of PTHrP (1–36) or PTHrP (107–139) was similarly efficient in improving the delayed regeneration of marrow-ablated mouse tibia in mice with glucocorticoid- or diabetic-related osteopenia [14,20,21]. The anabolic action of the N-terminal fragment 1–36 of PTHrP interacting with the common PTH/PTHrP type 1 receptor in osteoblasts is well characterized [22]. In contrast, the molecular basis of the observed osteogenic effects of C-terminal PTHrP (107–139) are ill-defined, although they might involve the interaction of its 107– 111 epitope (named osteostatin) with an as-yet uncharacterized receptor [23,24]. Interestingly, we recently showed that osteostatin-coated SBA15-based bioceramics exerted several osteogenic actions in osteoblastic cells in vitro as well as in a cavitary defect in the rabbit femur [25,26]. In the present study, we further evaluated the osteogenic properties of these osteostatin-loaded bioceramics by using a shorter time period than that used in our previous study after creating the above type of defect in healthy as well as in osteoporotic rabbits. This short time approach allowed us to monitor the differential occurrence of osteoblast precursors (using different osteoblast differentiation markers) in normal and osteopenic conditions. We also assessed the relative advantage of unmodified and organically modified SBA15 ceramics loaded with osteostatin to promote bone regeneration in this model.
Materials were exposed to UV irradiation for sterilization prior to surgery. 2.2. Experimental model Our protocol was designed to use a limited number of female New Zealand rabbits (24–30 weeks of age; n = 4 per experimental group) by means of multisite intervention, and was approved by the Institutional Animal Care and Use Committee at the Instituto de Investigación Sanitaria (IIS)-Fundación Jiménez Díaz, following the European Union guidelines for decreasing pain and suffering of animals. Rabbits were randomly used as healthy controls or assigned to osteoporosis induction as described previously [30]. For the latter, briefly, rabbits were daily injected with methylpredinisolone hemisuccinate (1.5 mg kg1 day1) for four consecutive weeks. Osteoporosis was confirmed by dual-energy X-ray absorptiometry (DEXA) analysis of bone mineral density (BMD) in both the lumbar spine and the left knee, performed in each rabbit using a Hologic QDR-1000/W pencil beam densitometer (Hologic, Waltham, MA, USA) with a 1 mm diameter collimator on the X-ray output, and using specific software for small animals (version 6.2). Thereafter, lateral and medial cavitary defects were created in the distal femoral epiphysis under general anesthesia. The surgical defect consisted of a 4 mm diameter cavity 3–4 mm deep, reamed in the metaphyseal bone after direct surgical approach. Care was taken to perform the reaming at low speed with sterile tips and sterile cover on the handle, using physiological serum to refrigerate bone during reaming of the cavity [26]. Unloaded and osteostatin-loaded SBA15 materials were then implanted into lateral and medial right femoral defects, respectively, whereas lateral and medial left femoral defects received unloaded and osteostatinloaded SBA15 + C8 materials, respectively. After that, the wounds were sutured. Post-surgery, the animals did not require immobilization and were allowed to move without restriction in the cage. Two weeks after implantation, animals were sacrificed under anesthesia, and both femora were removed for histological and immunohistochemical analysis. 2.3. Histological and immunohistochemical analysis
2. Materials and methods 2.1. Preparation of materials SBA15 material composed of 100% SiO2 was synthesized using a surfactant (PluronicÒ P123, BASF, New Jersey, USA) as structuredirecting agent and tetraethylorthosilicate (Sigma–Aldrich, St Louis, MO), following the procedure reported by Zhao et al. [27]. Its ordered mesoporous structure was confirmed by small angle X-ray diffraction in a Philips X’Pert MPD (Cu Ka radiation) diffractometer. The surface area and pore size of the materials were determined by N2 adsorption using a Micromeritics ASAP 2010 porosimeter. An organic modification of the silica walls was carried out by grafting an alkoxysilane, n-octyltriethoxysilane (C8, Sigma– Aldrich). This organic modification was carried out to change the polarity of the ceramic surface, resulting in both greater retention and slower release of drugs [25,28,29]. The grafting of the alkyl chain was confirmed by Fourier-transform infrared spectroscopy and quantified by CHN elemental analysis. For the experiments, both types of powders, functionalized and non-functionalized SBA15 materials, were conformed as 0.03 g disks (4 mm diameter 2 mm height) by uniaxial (1 MPa) and isostatic pressure (1 MPa). Loading with human PTHrP (107–111) amide (Bachem, Bubendorf, Switzerland) was performed by soaking the mesoporous materials in a solution of this peptide in phosphate-buffered saline (PBS), at 100 nM, for 24 h as recently described [23,24].
Femora were sequentially fixed in 10% neutral formaldehyde, decalcified with Osteosoft (Merck, Madrid, Spain), and dehydrated before paraffin embedding [26]. Histological analysis was performed on sagittal sections (5–8 lm) by hematoxylin/eosin and Masson’s staining, using light microscopy (Zeiss Axiophot; Carl Zeiss, Oberkochen, Germany). For immunohistochemistry, deparaffinized and rehydrated tissue specimens were pre-incubated in 4% bovine serum albumin in phosphate-buffered saline with 0.1% Triton X-100 for 30 min at room temperature. This was followed by incubation with the corresponding mouse monoclonal primary antibodies (dilution, fold): osteopontin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (50); osteocalcin antibody (Santa Cruz Biotechnology) (50); Runx2 antibody (Santa Cruz Biotechnology) (50); tartrate-resistant acid phosphatase (TRAP) antibody (Santa Cruz Biotechnology) (100); proliferating cell nuclear antigen (PCNA) antibody (ABCAM, Cambridge, UK) (50); rabbit monocyte/macrophage (RAM11) antibody (Dako, Barcelona, Spain) (50); and goat polyclonal anti-sclerostin antibody (R&D, Minneapolis, MN, USA) (500). Tissue samples were incubated with primary antibodies in a humidified chamber overnight or for 2 h at room temperature (for sclerostin). Sections were subsequently incubated with biotinlylated anti-goat IgG or horseradish peroxidase-coupled rabbit anti-goat IgG (sclerostin), followed by avidin-alkaline phosphatase and naphtol AS-BI phosphate as substrate. As chromogens, Fast Red or 3,30 -diaminobenzidine (sclerostin) were used. Sections were counterstained with haematoxylin.
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Fig. 1. Light microscopy from hematoxylin/eosin stained sections of the area surrounding SBA15 + C8 implant (m), alone (A) or containing osteostatin (B), in an osteoporotic rabbit at week 2 of bone regeneration in a femoral cavitary defect. Abundant newly formed bone (NB) was more apparent in the vicinity of the biomaterial (B) as compared to the plain (peptide unloaded) implant (A); FC = fibrous cap.
For each tested factor, cells showing immunostaining were counted in 10 consecutive 400-fields in the same area close to the implant. The resulting scores were expressed as the percentage of positive cells with respect to the total number of cells (showing positive and negative staining) in each field. Staining was evaluated by 2–3 independent observers in a blinded fashion and the corresponding mean score value was obtained for each animal. 2.4. Statistical analysis Results are expressed as mean ± SD throughout the text. Changes in the response to peptide coating between control and osteoporotic rabbits for each type of ceramic tested were assessed by non-parametric t-test (assuming equal variances) with statistical software GraphPad Prism 5.0. p < 0.05 was considered significant. 3. Results We did not observe any clinical alterations such as fever, infection, or behavioral parameters (appetite loss, irritability) as a
consequence of surgery or the type of implanted material in the rabbits. BMD measurements in the lumbar spine of osteoporotic rabbits, calculated by DEXA of L3 and L4, provided a value (g cm2; mean ± SD) of 264 ± 23. BMD measurements obtained from six regions of interest in the knee – distal femur and proximal tibia previously referred to as ‘‘global knee’’ BMD [30] – provided a value (mean ± SD) of 337 ± 19. These values are very similar to those recently reported for skeletally mature female rabbits with glucocorticoid-induced osteoporosis following a similar protocol to that used here [30]. Macroscopically, both types of plain (unloaded) ceramics were intact and encapsulated by fibrotic tissue within the cavitary defect, with no morphological evidence of inflammatory response. In the osteoporotic animals, granulated scar tissue was the main component of this fibrotic cap, which was somewhat smaller than that in healthy controls, while the callus close to the implant showed abundant mesenchymal cells and the presence of newly formed trabeculae. Osteostatin-loaded bioceramics promoted tissue regeneration mainly in osteoporotic rabbits. Abundant cubical cells and osteoid tissue make up the scar tissue surrounding these
Fig. 2. PCNA immunostaining in cells in the vicinity of the implanted material (m) in control and osteoporotic rabbits at 2 weeks. Corresponding score values (mean ± SD; n = 4) are shown. ⁄p < 0.01 vs. corresponding plain material. Representative images show the pattern of staining corresponding to SBA15 with and without osteostatin in a control and an osteoporotic animal, respectively.
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Fig. 3. Changes in sclerostin immunostaining in the area surrounding the different implants in a femoral defect in control and osteoporotic rabbits at 2 weeks. Corresponding score values (mean ± SD; n = 4) are shown.
biomaterials, while new trabeculae appeared more distant from the implant (Fig. 1). Of note, the fibrotic capsule around the SBA15 + C8 biomaterial was thinner and the osteogenic response appeared to be greater as compared to the SBA15 biomaterial, consistent with our previous findings [26]. PCNA positivity, denoting proliferative activity, was observed in the regenerating tissue around the implanted materials in healthy rabbits, but it was lower in osteoporotic rabbits (Fig. 2). This positivity significantly increased in the presence of osteostatin in these biomaterials in the former animals, and also in osteoporotic rabbits, but only those bearing the SBA15 biomaterial (Fig. 2). We also performed staining for the SOST gene product, sclerostin, which is specifically expressed in osteocytes [31], to evaluate osteocyte number in the area around the implants. We failed to find sclerostin positive osteocytes in any of the healthy groups of rabbits studied. In contrast, osteoporotic animals showed a small amount of sclerostin positive cells, with a tendency to increase in rabbits with an SBA15 + C8 implant. The presence of osteostatin did not significantly modify this pattern of staining (Fig. 3). Nuclear immunostaining for the osteogenic transcription factor Runx2 was higher in cells around both osteostatin-loaded biomaterials
tested as compared to the corresponding plain materials in healthy rabbits (Fig. 4). A similar trend, although somewhat attenuated, was observed in the pattern of staining for this factor in both osteoporotic groups (Fig. 4). Osteopontin is a major noncollagenous extracellular matrix protein in bone which modulates a variety of processes such as angiogenesis, mineralization and osteoclastic bone resorption [32]. We found that the number of osteopontin-positive cells was increased around both types of tested materials in the osteoporotic rabbits, mainly in those bearing the osteostatin-loaded SBA15 + C8 biomaterial, compared to that observed in their healthy controls (Fig. 5A and C). Meanwhile, the number of cells positive for osteocalcin – a late osteoblast differentiation marker – was unchanged in the callus in osteoporotic rabbits (Fig. 5B and D). At this early stage of bone regeneration, a low number of RAM11-positive cells were detected in the repairing area around the different implants in healthy rabbits (not shown) and in osteoporotic rabbits (Fig. 6A). TRAP positive multinuclear cells adjacent to new bone surfaces – likely osteoclasts – were similarly scarce in these rabbits at this time period (Fig. 6B). Interestingly, however, we found that the RAM11+/TRAP+ ratio in these animals bearing the SBA15 implant significantly decreased related to the local presence of osteostatin, similarly to the effect observed with the plain SBA15 + C8 implant (Fig. 6C). Angiogenesis is known to play an important role in bone regeneration. Consistent with our recent findings [26], the presence of osteostatin in both types of tested biomaterials dramatically increased the number of VEGF-positive cells in the regenerating tissue but only in healthy rabbits as opposed to osteoporotic rabbits (Fig. 7). 4. Discussion Recently, we demonstrated that osteostatin confers osteoconductive and osteoinductive features to SBA15-based ceramics when implanted into a cavitary bone defect in the rabbit femur [26]. In this previous study, we included both histological assessment and lCT to estimate new bone formation. In the present study, we focused on the early osteogenic stages (at 2 weeks) of this process, and thus we performed histology and immunohistochemistry evaluations which provide more specific data than standard radiologic methods in this setting. Using this shorter time period, we confirmed here our previous findings at 4 and 8 weeks in the same animal model [26]. Hence,
Fig. 4. Runx2 immunostaining in the implant–bone interface within a cavitary defect in the femur of control and osteoporotic rabbits (A). Corresponding score values (mean ± SD; n = 4) are shown. ⁄p < 0.01 vs. corresponding plain material. A representative image depicts abundant positivity for this transcription factor in the newly formed bone area in a control rabbit with osteostatin-containing SBA15 + C8 implant (B).
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Fig. 5. Changes in osteopontin (A) and osteocalcin (B) immunostaining in the implant–bone interface within the femoral defect in control and osteoporotic rabbits. Corresponding score values (mean ± SD; n = 4) are shown. ⁄p < 0.01 vs. corresponding plain material. Representative images show the pattern of staining (C and D) in an osteoporotic animal with SBA15 + C8 containing osteostatin.
Fig. 6. Changes in RAM11 (A) and TRAP (B) immunostaining as well as in RAM11/TRAP positivity ratio (C) in the peri-implant area at 2 weeks following a cavitary defect in osteoporotic rabbits. Corresponding score values (mean ± SD; n = 4) are shown. ⁄p < 0.05 vs. plain SBA15 material.
both osteostatin-containing SBA15 and SBA15 + C8 implants induced the recruitment of proliferating mesenchymal cells surrounding the fibrous cap – which was thinner with the latter implant – as well as abundant osteoprogenitors invading the implant border. These responses were more dramatic in osteoporotic rabbits, possibly related to the fact that the injured tissue appeared to be more reactive to the implant in these animals than in healthy controls. In addition, the present data further support the notion that administration of a low glucocorticoid dose (1.5 mg kg1 as daily injection) provides a suitable osteoporotic model [30].
In the present study, the local presence of osteostatin was shown to increase cell proliferation around both types of implanted biomaterials in control rabbits, whereas this osteostatinrelated effect was only significant in osteoporotic rabbits with SBA15 implant. Sclerostin-positive osteocytes in the newly formed bone were found only in osteoporotic rabbits, consistent with the inhibitory effect of this protein on bone formation [33]. The presence of osteostatin failed to affect this osteocyte staining, somewhat in contrast to what was observed during bone regeneration at a longer time (4–8 weeks) in our previous report [26]. Thus,
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Fig. 7. Changes in VEGF positivity in the regenerating femur close to the implanted material (m)-bone stroma interface in control and osteoporotic rabbits. Corresponding score values (mean ± SD; n = 4) are shown (A). ⁄p < 0.01 vs. corresponding plain material. Representative image depicts VEGF staining pattern in a control rabbit with SBA15 + osteostatin (B).
our data cannot rule out a direct modulatory effect of osteostatin on Sost/sclerostin in osteoporotic animals, as suggested by our recent in vitro data [34]. Runx2 positive cells increased early (at 2 weeks) in regenerating bone after implanting the osteostatin-containing bioceramics in normal rabbits, consistent with our recent observations in this model but at later time periods of bone repair [26]. Furthermore, our findings also indicate that this transcription factor can be a target of osteostatin in osteoporotic rabbits. In this regard, a previous report showed that the osteostatin-related peptide, PTHrP (107– 139), stimulated Runx2 expression and activity in both human osteoblastic cells in vitro and ovariectomized mice in vivo [34–36]. The osteogenic action of osteostatin at the early stage of bone repair was further confirmed by the observed increase in osteopontin staining in osteoporotic rabbits, considering the key role of this extracellular matrix component in early callus formation [37]. The lack of significant changes in osteocalcin staining in both groups of rabbits studied at 2 weeks of bone regeneration is not surprising since this is a marker for more differentiated osteoblasts. Our findings here support the notion that osteostatin stimulated the appearance of osteoblast precursors, based on PCNA, Runx2 and osteopontin positivity, in osteoporotic rabbits at the early stage of bone regeneration. The current hypothesis establishes that mesenchymal cells committed to the osteoblastic lineage may recruit osteoclast precursors and increase bone resorption during the first 1–3 weeks in the bone regeneration process [5,26]. In fact, the observed changes in RAM11/TRAP staining ratio within this time range in regenerating osteoporotic bone supports this concept. As expected, based on our recent report [26], both osteostatinloaded bioceramics increased VEGF immunostaining in the regenerating bone tissue in healthy rabbits at 2 weeks. However, this was not the case in osteoporotic rabbits at this time. Since both the VEGF system and vascularization are decreased in the setting of osteopenia [12,14,20,21], neovascularization in the callus is likely to be delayed in osteoporotic rabbits. Thus, the time period of this study might be too short for the angiogenic action of osteostatin to be apparent in these animals. 5. Conclusions These findings demonstrate that SBA15-based ceramics containing osteostatin as implants are biocompatible and induce
recruitment and activation of osteoprogenitors to promote bone regeneration in osteoporotic rabbits. Our data confirm that organically modified SBA15 ceramics can be advantageous in this setting, as they generate a thinner fibrous cap. These results strongly support the usefulness of these biomaterials as potential therapies to improve bone repair in osteoporotic subjects. Acknowledgements This paper is devoted to the memory of our colleague Prof. Purificación Escribano. We are grateful to Dr. S. Castañeda (Hospital La Princesa, Madrid) for performing BMD measurements, and to Mark Davis for proofreading the manuscript. This study was supported by Grants from the Spanish Instituto de Salud Carlos III (PI080922, and RETICEF RD06/0013/1002), Fundación de Investigación Médica Mutua Madrileña, Comisión Interministerial de Ciencia y Tecnología (CICYT, Spain) (MAT2008-736 and CSO2010-11384-E) and Comunidad Autónoma de Madrid (CAM; S2009/MAT-1472). D.L. was supported by Conchita Rábago Foundation and is currently the recipient of a post-doctoral research contract from CAM. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1, 2, 4, 5, and 7, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2012.03.014. References [1] Le AX, Miclau T, Hu D, Helms JA. Molecular aspects of healing in stabilized and non-stabilized fractures. J Orthop Res 2001;19:78–84. [2] Deschaseaux F, Sensébé L, Heymann D. Mechanisms of bone repair and regeneration. Trends Mol Med 2009;15:417–29. [3] Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem 2003;88:873–84. [4] Pederson L, Ruan M, Westendorf JJ, Khosla S, Oursler MJ. Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate. Proc Natl Acad Sci USA 2008;105:20764–9. [5] Caetano-Lopes J, Lopes A, Rodrigues A, Fernandes D, Perpétuo IP, Monjardino T, et al. Upregulation of inflammatory genes and downregulation of sclerostin gene expression are key elements in the early phase of fragility fracture healing. PLoS One 2011;6:e16947. [6] De Biase P, Capanna R. Clinical applications of BMPs. Injury 2005;36(Suppl. 3):S43–S466. [7] Kim J-B, Leucht P, Lam K, Luppen C, Berge DT, Nusse R, et al. Bone regeneration is regulated by Wnt signaling. J Bone Miner Res 2007;22:1913–23.
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1–34, parathyroid hormone-related protein 1–36, and SDZ-parathyroid hormone 893. J Bone Miner Res 2000;15:1517–25. Valín A, Guillén C, Esbrit P. C-terminal parathyroid hormone-related protein (PTHrP) (107–139) stimulates intracellular Ca2+ through a receptor different from the type 1 PTH/PTHrP receptor in osteoblastic osteosarcoma UMR 106 cells. Endocrinology 2001;142:2752–9. Cuthbertson RM, Kemp BE, Barden JA. Structure study of osteostatin PTHrP[Thr107](107–139). Biochim Biophys Acta 1999;1432:64–72. Lozano D, Manzano M, Doadrio JC, Salinas AJ, Vallet-Regí M, Gómez-Barrena E, et al. Osteostatin-loaded bioceramics stimulate osteoblastic growth and differentiation. Acta Biomater 2010;6:797–803. Trejo CG, Lozano D, Manzano M, Doadrio JC, Salinas AJ, Dapía S, et al. The osteoinductive properties of mesoporous silicate coated with osteostatin in a rabbit femur cavity defect model. Biomaterials 2010;31:8564–73. Zhao D, Hou Q, Fena J, Chmelka BF, Stucky GD. Nonionic triblock and star diblock copolymer and oligomeric surfactant synthesis of highly ordered, hydrothermally stable, mesoporous silica structures. J Am Chem Soc 1998;120:6024–36. Doadrio JC, Sousa EMB, Izquierdo-Barba I, Doadrio AL, Pérez-Pariente J, ValletRegí M. Functionalization of mesoporous materials with long alkyl chains as a strategy for controlling drug delivery pattern. J Mater Chem 2006;16:462–6. Doadrio AL, Doadrio JC, Sánchez-Montero JM, Salinas AJ, Vallet-Regí M. A rational explanation of the vancomycin release from SBA15 and its derivative by molecular modeling. Micropor Mesopor Mater 2010;132:559–66. Castañeda S, Calvo E, Largo R, González-González R, de la Piedra C, Díaz-Curiel M, et al. Characterization of a new experimental model of osteoporosis in rabbits. J Bone Miner Metab 2008;26:53–9. Portal-Núñez S, Lozano D, Fernández de Castro L, de Gortázar AR, Nogués X, Esbrit P. Alterations of the Wnt/b-catenin pathway and its target genes for the N- and C-terminal domains of parathyroid hormone-related protein in bone from diabetic mice. FEBS Lett 2010;584:3095–100. Alford AI, Hankenson KD. Matricellular proteins: extracellular modulators of bone development, remodeling, and regeneration. Bone 2006;38:749–57. Li X, Ominsky MS, Niu QT, Sun N, Daugherty B, D’Agostin D, et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res 2008;23:860–91. de Castro LF, Lozano D, Portal-Núñez S, Maycas M, De la Fuente M, Caeiro JR, et al. Comparison of the Skeletal effects induced by daily administration of PTHrP (1–36) and PTHrP (107–139) to ovariectomized mice. J Cell Physiol 2012;227:1752–60. Alonso V, de Gortázar AR, Ardura JA, Andrade-Zapata I, Álvarez-Arroyo MV, Esbrit P. Parathyroid hormone-related protein (107–139) increases human osteoblastic cell survival by activation of vascular endothelial growth factor receptor-2. J Cell Physiol 2008;217:717–27. de Gortázar AR, Alonso V, Alvarez-Arroyo MV. Esbrit. Transient exposure to PTHrP-107–139 exerts anabolic effects through vascular endothelial growth factor receptor 2 in human osteoblastic cells in vitro. Calcif Tissue Int 2006;79:360–9. Duvall CL, Taylor WR, Weiss D, Wojtowicz AM, Guldberg RE. Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice. J Bone Miner Res 2007;22:286–97.
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Osteostatin-loaded onto mesoporous ceramics improves the early phase of bone regeneration in a rabbit osteopenia model Daniel Lozano a,b,1, Cynthia G. Trejo c, Enrique Gómez-Barrena d,1, Miguel Manzano e,f, Juan C. Doadrio e,f, Antonio J. Salinas e,f, María Vallet-Regí e,f,⇑, Natalio García-Honduvilla c, Pedro Esbrit a,b, Julia Buján c a
Laboratorio de Metabolismo Mineral y Óseo, Instituto de Investigación Sanitaria (IIS)-Fundación Jiménez Díaz, 28040 Madrid, Spain Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF), Instituto de Salud Carlos III, 28029 Madrid, Spain Departamento de Especialidades Médicas, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain d Departamento de Cirugía Ortopédica y Traumatología, Hospital La Paz, Universidad Autónoma de Madrid, 28046 Madrid, Spain e Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain f Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain b c
a r t i c l e
i n f o
Article history: Received 16 November 2011 Received in revised form 1 March 2012 Accepted 6 March 2012 Available online 10 March 2012 Keywords: Osteoporosis Osteostatin Bone regeneration SBA15 mesoporous silica
a b s t r a c t Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Recently, we reported that PTHrP (107–111) (osteostatin) coating onto mesoporous ceramics confers osteogenic activity to these materials. Bone repair is dramatically compromised in osteopenia/osteoporosis. Thus, we examined the efficacy of unmodified and organically modified SBA15 ceramics loaded with osteostatin in promoting bone repair in an osteoporotic rabbit model. Osteoporosis was induced in New Zealand rabbits by methylprednisolone administration, and healthy rabbits were used as controls. Tested materials were implanted into a femoral cavitary defect, and animals were sacrificed at 2 weeks post-implantation. At this time, implants were encapsulated by a variable layer of fibrotic tissue with no evidence of inflammation. Similarly to observations in normal rabbits, both types of osteostatin-loaded bioceramics induced tissue regeneration associated with increased staining for PCNA, Runx2, osteopontin, and/or vascular endothelial growth factor in osteoporotic rabbits. Our present findings demonstrate that these osteostatinbearing bioceramics increase the early repair response not only in normal bone but also in osteoporotic bone after a local injury. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Bone repair to reconstitute skeletal integrity begins immediately after trauma. The healing events involve a hematoma-related infiltration of inflammatory cells, which increases the local delivery of cytokines at the site of damage, promoting both the recruitment and growth of mesenchymal stem cells from the periostial cambium [1,2]. These cells then respond by committing to the chondrogenic or osteogenic lineage, depending on whether vasculature is disrupted (close to the bone disjunction) or better preserved, respectively; the latter process is also favoured by the mechanical stability of the injured bone. Thus, the repair process is classically thought to involve both intramembranous and endochondral ossification followed by osteoclast-directed
⇑ Corresponding author at: Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. Tel.: +34 91 394 1861; fax: +34 91 394 1786. E-mail address: [email protected] (M. Vallet-Regí). 1 Present address: Grupo de Investigación de Cirugía OsteoArticular, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), 28046 Madrid, Spain.
remodelling [3]. Recent evidence also supports the notion that at an early stage during bone regeneration, recruitment of stromal cells may activate osteoclastogenesis and osteoclast-mediated resorption of damaged bone. This sets the conditions for new bone formation in a way that mimics bone remodelling within the basic multicellular units in the bone surface [4,5]. Knowledge of the molecular pathways and factors implicated in bone regeneration after skeletal trauma is still limited. However, the important roles played by bone morphogenetic proteins (BMPs), namely BMP-2 [2,6], and, as shown more recently, by Wnt/b-catenin pathway factors [7], in this regard provide a molecular rationale for using these factors as osteoinductive therapies. Injured bone tissue revascularization is a source of oxygen, nutrients and cell precursors, and thus is critical for successful bone repair. This is mainly carried out by the production of vascular endothelial growth factor (VEGF) in response to BMPs and other local factors in the callus, establishing the basis for VEGF administration as a putative therapy to enhance skeletal healing [8,9]. Other factors such as interleukins-1 and -6 and receptor activator of NF-jB ligand have an important role in osteoclast recruitment and newly formed bone remodelling [5,10].
1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.03.014
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Much less is currently known about the true underlying mechanisms of the observed reduction of bone repair in the setting of osteopenia. In this regard, an impaired fracture healing occurs with aging, which might be caused at least in part by a decrease in both osteogenic commitment of mesenchymal cells and angiogenesis [11,12]. Moreover, bone regeneration and bone repair have been shown to be dramatically hampered in diabetic conditions, associated with a decreased osteoblastic function [13,14]. This deficit can be reversed by systemic administration of insulin or parathyroid hormone (PTH)-related protein (PTHrP), an important modulator of bone formation and bone remodelling [14,15]. Moreover, this type of administration of PTH or PTHrP was shown to promote bone repair in ovariectomized rats [16–18] or rabbits with glucocorticoid-related osteopenia [19], respectively. Furthermore, it was recently shown that every other day injection of PTHrP (1–36) or PTHrP (107–139) was similarly efficient in improving the delayed regeneration of marrow-ablated mouse tibia in mice with glucocorticoid- or diabetic-related osteopenia [14,20,21]. The anabolic action of the N-terminal fragment 1–36 of PTHrP interacting with the common PTH/PTHrP type 1 receptor in osteoblasts is well characterized [22]. In contrast, the molecular basis of the observed osteogenic effects of C-terminal PTHrP (107–139) are ill-defined, although they might involve the interaction of its 107– 111 epitope (named osteostatin) with an as-yet uncharacterized receptor [23,24]. Interestingly, we recently showed that osteostatin-coated SBA15-based bioceramics exerted several osteogenic actions in osteoblastic cells in vitro as well as in a cavitary defect in the rabbit femur [25,26]. In the present study, we further evaluated the osteogenic properties of these osteostatin-loaded bioceramics by using a shorter time period than that used in our previous study after creating the above type of defect in healthy as well as in osteoporotic rabbits. This short time approach allowed us to monitor the differential occurrence of osteoblast precursors (using different osteoblast differentiation markers) in normal and osteopenic conditions. We also assessed the relative advantage of unmodified and organically modified SBA15 ceramics loaded with osteostatin to promote bone regeneration in this model.
Materials were exposed to UV irradiation for sterilization prior to surgery. 2.2. Experimental model Our protocol was designed to use a limited number of female New Zealand rabbits (24–30 weeks of age; n = 4 per experimental group) by means of multisite intervention, and was approved by the Institutional Animal Care and Use Committee at the Instituto de Investigación Sanitaria (IIS)-Fundación Jiménez Díaz, following the European Union guidelines for decreasing pain and suffering of animals. Rabbits were randomly used as healthy controls or assigned to osteoporosis induction as described previously [30]. For the latter, briefly, rabbits were daily injected with methylpredinisolone hemisuccinate (1.5 mg kg1 day1) for four consecutive weeks. Osteoporosis was confirmed by dual-energy X-ray absorptiometry (DEXA) analysis of bone mineral density (BMD) in both the lumbar spine and the left knee, performed in each rabbit using a Hologic QDR-1000/W pencil beam densitometer (Hologic, Waltham, MA, USA) with a 1 mm diameter collimator on the X-ray output, and using specific software for small animals (version 6.2). Thereafter, lateral and medial cavitary defects were created in the distal femoral epiphysis under general anesthesia. The surgical defect consisted of a 4 mm diameter cavity 3–4 mm deep, reamed in the metaphyseal bone after direct surgical approach. Care was taken to perform the reaming at low speed with sterile tips and sterile cover on the handle, using physiological serum to refrigerate bone during reaming of the cavity [26]. Unloaded and osteostatin-loaded SBA15 materials were then implanted into lateral and medial right femoral defects, respectively, whereas lateral and medial left femoral defects received unloaded and osteostatinloaded SBA15 + C8 materials, respectively. After that, the wounds were sutured. Post-surgery, the animals did not require immobilization and were allowed to move without restriction in the cage. Two weeks after implantation, animals were sacrificed under anesthesia, and both femora were removed for histological and immunohistochemical analysis. 2.3. Histological and immunohistochemical analysis
2. Materials and methods 2.1. Preparation of materials SBA15 material composed of 100% SiO2 was synthesized using a surfactant (PluronicÒ P123, BASF, New Jersey, USA) as structuredirecting agent and tetraethylorthosilicate (Sigma–Aldrich, St Louis, MO), following the procedure reported by Zhao et al. [27]. Its ordered mesoporous structure was confirmed by small angle X-ray diffraction in a Philips X’Pert MPD (Cu Ka radiation) diffractometer. The surface area and pore size of the materials were determined by N2 adsorption using a Micromeritics ASAP 2010 porosimeter. An organic modification of the silica walls was carried out by grafting an alkoxysilane, n-octyltriethoxysilane (C8, Sigma– Aldrich). This organic modification was carried out to change the polarity of the ceramic surface, resulting in both greater retention and slower release of drugs [25,28,29]. The grafting of the alkyl chain was confirmed by Fourier-transform infrared spectroscopy and quantified by CHN elemental analysis. For the experiments, both types of powders, functionalized and non-functionalized SBA15 materials, were conformed as 0.03 g disks (4 mm diameter 2 mm height) by uniaxial (1 MPa) and isostatic pressure (1 MPa). Loading with human PTHrP (107–111) amide (Bachem, Bubendorf, Switzerland) was performed by soaking the mesoporous materials in a solution of this peptide in phosphate-buffered saline (PBS), at 100 nM, for 24 h as recently described [23,24].
Femora were sequentially fixed in 10% neutral formaldehyde, decalcified with Osteosoft (Merck, Madrid, Spain), and dehydrated before paraffin embedding [26]. Histological analysis was performed on sagittal sections (5–8 lm) by hematoxylin/eosin and Masson’s staining, using light microscopy (Zeiss Axiophot; Carl Zeiss, Oberkochen, Germany). For immunohistochemistry, deparaffinized and rehydrated tissue specimens were pre-incubated in 4% bovine serum albumin in phosphate-buffered saline with 0.1% Triton X-100 for 30 min at room temperature. This was followed by incubation with the corresponding mouse monoclonal primary antibodies (dilution, fold): osteopontin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (50); osteocalcin antibody (Santa Cruz Biotechnology) (50); Runx2 antibody (Santa Cruz Biotechnology) (50); tartrate-resistant acid phosphatase (TRAP) antibody (Santa Cruz Biotechnology) (100); proliferating cell nuclear antigen (PCNA) antibody (ABCAM, Cambridge, UK) (50); rabbit monocyte/macrophage (RAM11) antibody (Dako, Barcelona, Spain) (50); and goat polyclonal anti-sclerostin antibody (R&D, Minneapolis, MN, USA) (500). Tissue samples were incubated with primary antibodies in a humidified chamber overnight or for 2 h at room temperature (for sclerostin). Sections were subsequently incubated with biotinlylated anti-goat IgG or horseradish peroxidase-coupled rabbit anti-goat IgG (sclerostin), followed by avidin-alkaline phosphatase and naphtol AS-BI phosphate as substrate. As chromogens, Fast Red or 3,30 -diaminobenzidine (sclerostin) were used. Sections were counterstained with haematoxylin.
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Fig. 1. Light microscopy from hematoxylin/eosin stained sections of the area surrounding SBA15 + C8 implant (m), alone (A) or containing osteostatin (B), in an osteoporotic rabbit at week 2 of bone regeneration in a femoral cavitary defect. Abundant newly formed bone (NB) was more apparent in the vicinity of the biomaterial (B) as compared to the plain (peptide unloaded) implant (A); FC = fibrous cap.
For each tested factor, cells showing immunostaining were counted in 10 consecutive 400-fields in the same area close to the implant. The resulting scores were expressed as the percentage of positive cells with respect to the total number of cells (showing positive and negative staining) in each field. Staining was evaluated by 2–3 independent observers in a blinded fashion and the corresponding mean score value was obtained for each animal. 2.4. Statistical analysis Results are expressed as mean ± SD throughout the text. Changes in the response to peptide coating between control and osteoporotic rabbits for each type of ceramic tested were assessed by non-parametric t-test (assuming equal variances) with statistical software GraphPad Prism 5.0. p < 0.05 was considered significant. 3. Results We did not observe any clinical alterations such as fever, infection, or behavioral parameters (appetite loss, irritability) as a
consequence of surgery or the type of implanted material in the rabbits. BMD measurements in the lumbar spine of osteoporotic rabbits, calculated by DEXA of L3 and L4, provided a value (g cm2; mean ± SD) of 264 ± 23. BMD measurements obtained from six regions of interest in the knee – distal femur and proximal tibia previously referred to as ‘‘global knee’’ BMD [30] – provided a value (mean ± SD) of 337 ± 19. These values are very similar to those recently reported for skeletally mature female rabbits with glucocorticoid-induced osteoporosis following a similar protocol to that used here [30]. Macroscopically, both types of plain (unloaded) ceramics were intact and encapsulated by fibrotic tissue within the cavitary defect, with no morphological evidence of inflammatory response. In the osteoporotic animals, granulated scar tissue was the main component of this fibrotic cap, which was somewhat smaller than that in healthy controls, while the callus close to the implant showed abundant mesenchymal cells and the presence of newly formed trabeculae. Osteostatin-loaded bioceramics promoted tissue regeneration mainly in osteoporotic rabbits. Abundant cubical cells and osteoid tissue make up the scar tissue surrounding these
Fig. 2. PCNA immunostaining in cells in the vicinity of the implanted material (m) in control and osteoporotic rabbits at 2 weeks. Corresponding score values (mean ± SD; n = 4) are shown. ⁄p < 0.01 vs. corresponding plain material. Representative images show the pattern of staining corresponding to SBA15 with and without osteostatin in a control and an osteoporotic animal, respectively.
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Fig. 3. Changes in sclerostin immunostaining in the area surrounding the different implants in a femoral defect in control and osteoporotic rabbits at 2 weeks. Corresponding score values (mean ± SD; n = 4) are shown.
biomaterials, while new trabeculae appeared more distant from the implant (Fig. 1). Of note, the fibrotic capsule around the SBA15 + C8 biomaterial was thinner and the osteogenic response appeared to be greater as compared to the SBA15 biomaterial, consistent with our previous findings [26]. PCNA positivity, denoting proliferative activity, was observed in the regenerating tissue around the implanted materials in healthy rabbits, but it was lower in osteoporotic rabbits (Fig. 2). This positivity significantly increased in the presence of osteostatin in these biomaterials in the former animals, and also in osteoporotic rabbits, but only those bearing the SBA15 biomaterial (Fig. 2). We also performed staining for the SOST gene product, sclerostin, which is specifically expressed in osteocytes [31], to evaluate osteocyte number in the area around the implants. We failed to find sclerostin positive osteocytes in any of the healthy groups of rabbits studied. In contrast, osteoporotic animals showed a small amount of sclerostin positive cells, with a tendency to increase in rabbits with an SBA15 + C8 implant. The presence of osteostatin did not significantly modify this pattern of staining (Fig. 3). Nuclear immunostaining for the osteogenic transcription factor Runx2 was higher in cells around both osteostatin-loaded biomaterials
tested as compared to the corresponding plain materials in healthy rabbits (Fig. 4). A similar trend, although somewhat attenuated, was observed in the pattern of staining for this factor in both osteoporotic groups (Fig. 4). Osteopontin is a major noncollagenous extracellular matrix protein in bone which modulates a variety of processes such as angiogenesis, mineralization and osteoclastic bone resorption [32]. We found that the number of osteopontin-positive cells was increased around both types of tested materials in the osteoporotic rabbits, mainly in those bearing the osteostatin-loaded SBA15 + C8 biomaterial, compared to that observed in their healthy controls (Fig. 5A and C). Meanwhile, the number of cells positive for osteocalcin – a late osteoblast differentiation marker – was unchanged in the callus in osteoporotic rabbits (Fig. 5B and D). At this early stage of bone regeneration, a low number of RAM11-positive cells were detected in the repairing area around the different implants in healthy rabbits (not shown) and in osteoporotic rabbits (Fig. 6A). TRAP positive multinuclear cells adjacent to new bone surfaces – likely osteoclasts – were similarly scarce in these rabbits at this time period (Fig. 6B). Interestingly, however, we found that the RAM11+/TRAP+ ratio in these animals bearing the SBA15 implant significantly decreased related to the local presence of osteostatin, similarly to the effect observed with the plain SBA15 + C8 implant (Fig. 6C). Angiogenesis is known to play an important role in bone regeneration. Consistent with our recent findings [26], the presence of osteostatin in both types of tested biomaterials dramatically increased the number of VEGF-positive cells in the regenerating tissue but only in healthy rabbits as opposed to osteoporotic rabbits (Fig. 7). 4. Discussion Recently, we demonstrated that osteostatin confers osteoconductive and osteoinductive features to SBA15-based ceramics when implanted into a cavitary bone defect in the rabbit femur [26]. In this previous study, we included both histological assessment and lCT to estimate new bone formation. In the present study, we focused on the early osteogenic stages (at 2 weeks) of this process, and thus we performed histology and immunohistochemistry evaluations which provide more specific data than standard radiologic methods in this setting. Using this shorter time period, we confirmed here our previous findings at 4 and 8 weeks in the same animal model [26]. Hence,
Fig. 4. Runx2 immunostaining in the implant–bone interface within a cavitary defect in the femur of control and osteoporotic rabbits (A). Corresponding score values (mean ± SD; n = 4) are shown. ⁄p < 0.01 vs. corresponding plain material. A representative image depicts abundant positivity for this transcription factor in the newly formed bone area in a control rabbit with osteostatin-containing SBA15 + C8 implant (B).
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Fig. 5. Changes in osteopontin (A) and osteocalcin (B) immunostaining in the implant–bone interface within the femoral defect in control and osteoporotic rabbits. Corresponding score values (mean ± SD; n = 4) are shown. ⁄p < 0.01 vs. corresponding plain material. Representative images show the pattern of staining (C and D) in an osteoporotic animal with SBA15 + C8 containing osteostatin.
Fig. 6. Changes in RAM11 (A) and TRAP (B) immunostaining as well as in RAM11/TRAP positivity ratio (C) in the peri-implant area at 2 weeks following a cavitary defect in osteoporotic rabbits. Corresponding score values (mean ± SD; n = 4) are shown. ⁄p < 0.05 vs. plain SBA15 material.
both osteostatin-containing SBA15 and SBA15 + C8 implants induced the recruitment of proliferating mesenchymal cells surrounding the fibrous cap – which was thinner with the latter implant – as well as abundant osteoprogenitors invading the implant border. These responses were more dramatic in osteoporotic rabbits, possibly related to the fact that the injured tissue appeared to be more reactive to the implant in these animals than in healthy controls. In addition, the present data further support the notion that administration of a low glucocorticoid dose (1.5 mg kg1 as daily injection) provides a suitable osteoporotic model [30].
In the present study, the local presence of osteostatin was shown to increase cell proliferation around both types of implanted biomaterials in control rabbits, whereas this osteostatinrelated effect was only significant in osteoporotic rabbits with SBA15 implant. Sclerostin-positive osteocytes in the newly formed bone were found only in osteoporotic rabbits, consistent with the inhibitory effect of this protein on bone formation [33]. The presence of osteostatin failed to affect this osteocyte staining, somewhat in contrast to what was observed during bone regeneration at a longer time (4–8 weeks) in our previous report [26]. Thus,
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Fig. 7. Changes in VEGF positivity in the regenerating femur close to the implanted material (m)-bone stroma interface in control and osteoporotic rabbits. Corresponding score values (mean ± SD; n = 4) are shown (A). ⁄p < 0.01 vs. corresponding plain material. Representative image depicts VEGF staining pattern in a control rabbit with SBA15 + osteostatin (B).
our data cannot rule out a direct modulatory effect of osteostatin on Sost/sclerostin in osteoporotic animals, as suggested by our recent in vitro data [34]. Runx2 positive cells increased early (at 2 weeks) in regenerating bone after implanting the osteostatin-containing bioceramics in normal rabbits, consistent with our recent observations in this model but at later time periods of bone repair [26]. Furthermore, our findings also indicate that this transcription factor can be a target of osteostatin in osteoporotic rabbits. In this regard, a previous report showed that the osteostatin-related peptide, PTHrP (107– 139), stimulated Runx2 expression and activity in both human osteoblastic cells in vitro and ovariectomized mice in vivo [34–36]. The osteogenic action of osteostatin at the early stage of bone repair was further confirmed by the observed increase in osteopontin staining in osteoporotic rabbits, considering the key role of this extracellular matrix component in early callus formation [37]. The lack of significant changes in osteocalcin staining in both groups of rabbits studied at 2 weeks of bone regeneration is not surprising since this is a marker for more differentiated osteoblasts. Our findings here support the notion that osteostatin stimulated the appearance of osteoblast precursors, based on PCNA, Runx2 and osteopontin positivity, in osteoporotic rabbits at the early stage of bone regeneration. The current hypothesis establishes that mesenchymal cells committed to the osteoblastic lineage may recruit osteoclast precursors and increase bone resorption during the first 1–3 weeks in the bone regeneration process [5,26]. In fact, the observed changes in RAM11/TRAP staining ratio within this time range in regenerating osteoporotic bone supports this concept. As expected, based on our recent report [26], both osteostatinloaded bioceramics increased VEGF immunostaining in the regenerating bone tissue in healthy rabbits at 2 weeks. However, this was not the case in osteoporotic rabbits at this time. Since both the VEGF system and vascularization are decreased in the setting of osteopenia [12,14,20,21], neovascularization in the callus is likely to be delayed in osteoporotic rabbits. Thus, the time period of this study might be too short for the angiogenic action of osteostatin to be apparent in these animals. 5. Conclusions These findings demonstrate that SBA15-based ceramics containing osteostatin as implants are biocompatible and induce
recruitment and activation of osteoprogenitors to promote bone regeneration in osteoporotic rabbits. Our data confirm that organically modified SBA15 ceramics can be advantageous in this setting, as they generate a thinner fibrous cap. These results strongly support the usefulness of these biomaterials as potential therapies to improve bone repair in osteoporotic subjects. Acknowledgements This paper is devoted to the memory of our colleague Prof. Purificación Escribano. We are grateful to Dr. S. Castañeda (Hospital La Princesa, Madrid) for performing BMD measurements, and to Mark Davis for proofreading the manuscript. This study was supported by Grants from the Spanish Instituto de Salud Carlos III (PI080922, and RETICEF RD06/0013/1002), Fundación de Investigación Médica Mutua Madrileña, Comisión Interministerial de Ciencia y Tecnología (CICYT, Spain) (MAT2008-736 and CSO2010-11384-E) and Comunidad Autónoma de Madrid (CAM; S2009/MAT-1472). D.L. was supported by Conchita Rábago Foundation and is currently the recipient of a post-doctoral research contract from CAM. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1, 2, 4, 5, and 7, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2012.03.014. References [1] Le AX, Miclau T, Hu D, Helms JA. Molecular aspects of healing in stabilized and non-stabilized fractures. J Orthop Res 2001;19:78–84. [2] Deschaseaux F, Sensébé L, Heymann D. Mechanisms of bone repair and regeneration. Trends Mol Med 2009;15:417–29. [3] Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem 2003;88:873–84. [4] Pederson L, Ruan M, Westendorf JJ, Khosla S, Oursler MJ. Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate. Proc Natl Acad Sci USA 2008;105:20764–9. [5] Caetano-Lopes J, Lopes A, Rodrigues A, Fernandes D, Perpétuo IP, Monjardino T, et al. Upregulation of inflammatory genes and downregulation of sclerostin gene expression are key elements in the early phase of fragility fracture healing. PLoS One 2011;6:e16947. [6] De Biase P, Capanna R. Clinical applications of BMPs. Injury 2005;36(Suppl. 3):S43–S466. [7] Kim J-B, Leucht P, Lam K, Luppen C, Berge DT, Nusse R, et al. Bone regeneration is regulated by Wnt signaling. J Bone Miner Res 2007;22:1913–23.
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