( PDF ) Rev Osteoporos Metab Miner. 2012; 4 (4): 117-26

Lozano D1, Trejo CG2, Manzano M3, Doadrio JC3, Salinas AJ3, Dapía S4, Caeiro Rey JR5, Gómez-Barrena E6, Vallet-Regí M3, García-Honduvilla N2, Buján J2, Esbrit P1
1 Laboratorio de Metabolismo Mineral y Óseo – Instituto de Investigación Sanitaria-Fundación Jiménez Díaz – Madrid
2 Departamento de Especialidades Médicas – Facultad de Medicina – Universidad de Alcalá – Alcalá de Henares – Madrid
3 Departamento de Química Inorgánica y Bioinorgánica – Facultad de Farmacia – Universidad Complutense – Madrid
4 Trabeculae S.L. – Orense
5 Complexo Hospitalario Universitario de Santiago de Compostela (CHUS) – Reticef – Santiago de Compostela
6 Departamento de Traumatología – Instituto de Investigación Sanitaria-Fundación Jiménez Díaz – Madrid

 

Summary
Introducción: Parathyroid hormone-related protein (PTHrP), which is abundant in bone tissue, is an important modulator of bone formation. It has been shown that PTHrP (107-111), called osteostatin, loaded into mesoporous ceramic material SBA-15 exerts osteogenic action in vitro.
Objective: To confirm if this material and a functionalised version of the same material (C8-SBA-15) promote bone regeneration in a model of a cavitary defect in a rabbit femur.
Materials and methods: Histological, immunohistochemical and computerised microtomography (µCT) studies were carried out in order to achieve the aims of the study.
Results: After the implantation of the biomaterials no significant levels of inflammation or bone resorption were observed (at 4 and 8 weeks). At 8 weeks the bioceramics not loaded with osteostatin were found to be separated from the bone medulla by a fibrous capsule which diminished significantly in the presence of the peptide. An increase was observed (using µCT) in bone neo-formation at different distances from the biomaterials, principally in those loaded with the osteostatin. These results were also confirmed by immunohistochemistry of osteoblast markers.
Conclusion: Our results suggest that the use of these osteostatin-loaded bioceramics are a good strategy for accelerating bone regeneration.
Keywords: PTHrP, osteostatin, bioceramics, bone regeneration, Introduction cavitary defect.

The protein related to parathormone (PTHrP) was initially identified in tumourous hypercalcemia of humeral origin1. Currently, it is known that PTHrP and the type 1 receptor common to PTH/PTHrP (PTHR1) is expressed in a wide variety of tissues, malignant and non-malignant, in which PTHrP exerts auto/paracrine and intracrine effects2. Although the fragment 1-36 of PTHrP, which has a structural homology with PTH, exerts anabolic actions on bone, stimulating bone formation3-6, the possible mechanisms associated with this action are little understood. The C-terminal fragment of PTHrP is a powerful inhibitor of osteoclast activity7-10. In fact, the pentapeptide 107-111 of this protein (called osteostatin) has a powerful antiresorptive activity in vitro and in vivo [11]. Various in vitro studies have shown that osteostatin increases the differentiation of osteoblast cells in mice and in humans12-15, although its receptor in bone cells is not known9,16. A study made by our laboratory has shown that the native fragment of PTHrP which contains the sequence 107-139 rapidly transactivates the receptor 2 for the vascular endothelial growth factor (VEGF) in osteoblast cells17. On the other hand, it has been shown that the daily administration of this fragment of PTHrP over a period of two weeks in ovariectomised mice protects against bone loss observed in their large bones, with positive effects in cortical and trabecular bone3. Recently, our group has also shown that PTHrP (107-139) reverses osteopenia and increases the regeneration of bone in mice treated with 3-methylprednisolone or those which are diabetic18,19.
An understanding the regeneration mechanisms is fundamental to understanding the behaviour of bone tissue after the implant of a prosthesis or during the recuperation from a fracture. It is known that the process of bone repair in adults reproduces the normal development of the skeleton during embryogenesis20. Bone formation in the foetus starts with the condensation of mesenchymal cells followed by their differentiation to chondrocytes (endochondral ossification) or directly to osteoblasts (intramembranous bone formation). In the majority of fractures, the formation of the callus involves a combination of both types of ossification. Although the signalling pathways or the cellular interactions involved in the repair of bone are not completely known, there are different strategies to improve this process20. One of these involves the systemic or local application of osteogenic factors which increase the formation of fresh bone. It has been shown that daily injections of PTH improve the repair of bone in fracture in normal or ovariectomised rats21,22. In addition, the systemic administration of an analogue to PTHrP (1-34) counteracts the deleterious effects in a defect in the ulna of rabbits treated with prednisolone23. A more direct approach to increase bone repair would consist of a local release of osteogenic factors with biomaterials in the affected zone.
Biomaterials may be defined as “implantable materials which develop their function in contact with living tissues”24. Recently, bioactive mesoporous ceramics have been designed which allow the adsorption and release of different molecules, with good prospects for clinical application25. The SBA (Santa Barbara Amorphous)-15 has a hexagonal structure of porous cylinders with a diameter of 5-10 nm. An advantage of this type of ceramic is its great porous volume and his concentration of SiOH groups26-28. In addition, this material has a series of interconnected micropores which promote greater ionic diffusion triggering a stronger bioactive response. In fact, the capture and release of L-tryptophan loaded into the SBA-15 material, and in a functionalised version (C8-SBA-15), has been studied [29]. The hydrophobic surface of the C8-SBA-15 material is capable of interacting with the indole ring of the tryptophan of different peptides and leaving smaller available space, occupied by alkyl chains. Our group has characterised the capture/release of osteocalcin loaded into these materials, demonstrating that the peptide confers on them osteogenic activity in osteoblasts in mice14.
The aim of the current study was to evaluate the capacity of the ceramic material SBO-15, functionalised or not with C8 groups (C8-SBA-15), loaded with PTHrP (107-111), to induce osteogenesis in a model of a cavity defect in a rabbit femur.

Materials and methods
Preparation of the materials
The mesoporous material SBA-15 was synthesised using a method based on the use of a surfactant, as a directing agent for the structure, and tetraethyl orthosilicate (Sigma-Aldrich, St Louis, MO) as the source of the silica35. This structure was confirmed by X-ray diffraction (XRD) and N2 adsorption analysis. The functionalisation of the silica was achieved using the post-synthetic method or anchorage grafting of an alkoxysilane, N-octyltriethoxysilane (C8, Sigma-Aldrich), as described30. This was confirmed using Fourier transform infrared electroscopy for elementary analysis. The resulting functionalised material contains 11% by weight (0.97 mmol/g) of organic fractions, which allows the calculation of the degree of functionalisation.
For the experiments both types of SBA-15 material were formed into 50 mg discs (6 x 2 mm) by uniaxial (1MPa) and isostatic (1MPa) pressure. The materials were exposed to ultraviolet radiation in a cell culture chamber (FLV120. Technology for Diagnosis and Research, Madrid), overnight for their sterilisation. The bond between the PTHrP (107-139) (Bacjchem, Bubendorf, Switzerland) and the material was made by immersion in a peptide solution (100nM) in 1 ml of saline phosphate buffer (SPB), pH 7.4 at 4° C with agitation for 24 hours31,32.

Production of the rabbit cavity defect
White male New Zealand rabbits were used (Granja San Bernardo, Valencia, Spain) aged 24-30 weeks [n=2 for computerised microtomography (µCT) and 5 for histological studies]. These animals were housed individually in cages in the animal facilities of the Jiménez Díaz Foundation over a period of two weeks. The animals had free access to water and a standard diet (Panlab, Reus, Spain) in a room kept at an ambient temperature with cycles of 12 hours of light and 12 hours of darkness. All the studies were carried out with the approval of the animal research committee of the Jiménez Díaz Foundation. The pain and suffering was minimised in accord with European rules.
The surgical intervention was carried out under general anaesthetic. The rabbit bone was shaved in both knees with a medium velocity drill (5 mm in diameter and 4-5 mm in depth) to create lateral and medial cavity defects33. Following this, the trial materials were implanted and the wounds sutured. The right lateral defect received the SBA-15 material without the peptide, and the right medial defect, the SBA-15 loaded with PTHrP (107-111). The left lateral femoral defect received the SBA-15 material functionalised with C8, while the biomaterial C8-SBA-15 loaded with PTHrP (107-111) was implanted in the left medial femoral defect. The animals were sacrificed at 4 and 8 weeks after these interventions. The femurs were distributed for histological and immunohistochemical tests and µCT analysis.

Histology
The rabbit femurs were fixed in p-formaldehyde at 4% in SPB at 4° C. The samples were decalcified (24 h) in Osteosoft (48/72 h) (Merk, Whitehouse Station, N.J.), dehydrated and embedded in paraffin. The histological analyses were carried out in sections of 8 µm on a sagittal plane, deposited on preprepared slides with L-Lysine (Polylysine, Thermo, Watham, MA), and then stained with haematoxylin/eosin. Before the staining the samples were kept at 60° C for 6-24 hours to fix the tissues to the slides. They were deparaffinised by incubating them sequentially in xylol, ethanol at 100%, and 70%, and distilled water. After staining the samples were dehydrated and mounted in DPX resin (a mixture of distyrene, plasticiser and xylol). Two histological sections from each rabbit were used from a total of 2-4 rabbits per experimental group. All the evaluations of the samples were analysed by 3 independent observers.

Immunohistochemistry
The histological slices obtained from the bone samples were deparaffinised and rehydrated. Blocking and permeablisation in bovine blood albumen at 4% (in SPB with 0.1% of Triton X-100) was carried out for 30 minutes at room temperature. Antibodies were used against osteocalcin (OC) (Santa Cruz Biotechnology, Santa Cruz, CA), VEGF (Abcam, Cambridge, MA) RAM11 (Dako) and TRAP (Santa Cruz Biotechnology), and a polyclonal antibody against sclerostin (R&D, Minneapolis, MN). The primary antibodies were incubated in a humidity chamber throughout the night at 4°C, except in the case of sclerostin which was incubated for two hours at room temperature (RT). In all the antibodies, except that for sclerostin, a secondary biotinylated antibody was used. It was incubated with alkaline extravidin-phosphatase complex (dilution 1:200), for 60 minutes at RT and washed with 3 times with SPB for 5 minutes. In the case of sclerostin the secondary antibody was bonded with peroxidise, which was incubated for 1 hour at RT. Development was carried out by incubation with the chromogen substrate DAB for 10 minutes. A sample without primary antibody was always included as a negative control. The samples were contrasted with haematoxylin. The cells positive for the different antibodies were determined in 10 fields in the vicinity of the material. In the case of the osteocytes positive for sclerostin, these were quantified in 5 random fields in the cortical bone.

Analysis of µCT
The samples of rabbit femur were cut with a hyperflexible disc of fine granulometry (15µm) and smooth abrasion connected to a surgical motor (KaVo, Dental GmbH, Biberach, Germany) at a velocity of 15,000 rpm. During the cutting process the sample was constantly irrigated with saline solution to prevent overheating and dehydration. The samples were scanned with a high resolution microtomography system (SkyScan 1172, Skyscan N.V., Aartselaar, Belgium), with an X-ray tube with a voltage of 100kV and a current of 100µA without filter. Once scanned, the images were generated using the DataViewer application (SkyScan), which were reconstructed according to the Feldkamp algorithm34. The angle of rotation of the scan was 360°. For the quantitative and qualitative analysis of bone growth around the implant the CTAn (SkyScan) application was used. From each sample a total of 70 images were analysed. The trabecular parameters were calculated in selected regions (Figure 1) between 0 and 5 pixels and 10 and 15 pixels, 1 pixel being the equivalent of 21.8 µm. Using the CTVol application (SkyScan) three-dimensional models were created both of the biomaterial (which was considered to be a cylinder for better clarity of presentation) and the bone formed at different distances from the surface of the implant. The system of analysis provided the calculations of the following trabecular parameters:
• Percentage of bone volume (BV/TV): relates the volume of calcified bone tissue with respect to the total volume of the area analysed (%).
• Trabecular bone pattern factor (Tb,Pf): index of connectivity of trabecular bone. This is based on the principle that a greater trabecular concavity indicates greater connectivity, by increasing the probability of connection nodes between the trabeculae. Thus a lower Tb.Pf indicates a higher trabecular connectivity (mm-1).

Statistical analysis
The results are expressed as mean ± standard error of the mean (SEM). The non-parametric comparison between two samples were carried out using the Mann Whitney test. Non-parametric ANOVA was used to compare various samples (Kruskal-Wallis) followed by a post-hoc test (Dunn). All values where p<0.05 were considered significant.

Results
Histological techniques and µCT were used to confirm the response of the tissue to the biomaterial and the effect of the PTHrP (107-111) on the cavity defect in the rabbit femur, at 4 and 8 weeks.
From the histopathological analysis of the bone samples the following results were obtained:

SBA-15
At 4 weeks from the implanting of the SBA-15 material, this was found to be intact, filling the cavity defect produced in the femur. The entry zone of the biomaterial was occupied by abundant dense connective tissue. Around the biomaterial was observed a large amount of osteoid (Figure 2A) in the cortical zones, and a fibrous capsule which isolated and enveloped the material, without apparently interfering with the medullar area. No inflammatory reaction was observed. At 8 weeks, the biomaterial in contact with the bone medulla did not have significant modifications, remaining isolated by the fibrous capsule. In addition, in the zone in contact with the cortical bone the formation of trabeculae was observed in the vicinity of the implant (Figure 4A).
C8-SBA-15
In the animals implanted with this material a similar behaviour was observed to that described for the implant of non-functionalised SBA-15 after 4 weeks. However, the C8-SBA-15 was seen to be isolated by a fibrous capsule of greater thickness (Figure 2C). The bone repair seen at 8 weeks covered the whole cavity defect, with a well defined external fibrous capsule being observed surrounded by osteoid which occupied the entry space of the cavity of the biomaterial and newly-formed bone trabeculae surrounding the cortical face of the biomaterial (Figure 4C).

SBA-15 + PTHrP (107-111)
At 4 weeks from the implant of the SBA-15 loaded with PTHrP (107-111) an intense scarring was observed which extended from the cavity to the entry of the biomaterial (now occupied by a thick layer of fibrous connective and articular cartilaginous tissue) with a great quantity of osteoid around it. The biomaterial in contact with the medullar area was seen to be surrounded by trabecular bone situated on the periphery of the osteoid surface (Figure 2B). On the other hand the osteoid described increased significantly around the cortical face of the biomaterial in the direction of the external surface at 8 weeks, while being, on the contrary, more limited towards the medullar zone where trabecular areas surrounding the implanted biomaterial were observed (Figure 4B).

C8-SBA-15 + PTHrP (107-111)
The behaviour of the C8-SBA-15 loaded with PTHrP (107-111) was similar to that described in the last group, the fibrous capsule maintaining a greater thickness in comparison with the groups not functionalised with C8 (Figure 2D). At 8 weeks, during the process of repair, external connective hyperplasia, and hyperplasia of the hyaline cartilage, was observed, as well as bone hyperplasia which formed the tissue layer of the osteoid neoformations surrounding the implant. Most notable in this group was the internal reaction observed in the fibrous capsule, which appeared to be split, allowing the appearance of a space occupied by non-calcified osteoid which outlined the intact surface of the biomaterial (Figure 4D).
The observations carried out in the histological study at 4 and 8 weeks for each of the biomaterials were confirmed by µCT studies. In the representative images of each experimental group it is possible to see the bone formation again, with the presence of trabeculae, at different distances from the implanted material (Figures 3 and 5). The increase in bone formation observed in the images corresponding to the materials loaded with PTHrP (107-111) is evident from the increase in the BV/TV % and the reduction in Tb.Pf in the rabbit femur (Table 1).
The changes observed in the histology and the bone structure of the rabbit femur were correlated with a significant increase in staining of OC in the samples with materials loaded with PTHrP (107-111) (Figure 6A). A significant increase was observed in the staining of femurs which contained the biomaterials loaded with PTHrP (107-111) in comparison with those which did not contain the peptide. It is especially worth noting that the presence of the peptide in the biomaterial C8-SBA-15 induced a greater effect in this marker than in the SBA-15 (Figure 6A). In none of the femurs studied was observed an increase in the inflammatory component in the presence of the different materials studied.
What is more, the presence of cells positive for the TRAP or RAM11 immunostain (Figures 6B and C) (macrophage marking) was very low in all the study groups (below 3%), and practically zero in the case of the materials with PTHrP (107-111). It was not possible to quantify the differences in positive immunostaining for VEGF (Figure 6D), since the marking was very faint for this marker in most of the study groups.

Discussion
The results described above demonstrate the osteogenic capacity of osteostatin, loaded into ceramic materials SBA-15 and C8-SBA-15, in a bone regeneration model. These findings open new perspectives in the context of bone repair, since they suggest that local exposure to this pentapeptide in the bone environment would promote its regeneration. Recently, our group has demonstrated the capacity of the materials SBA-15 and C8-SBA-15 with PTHrP (107-111) to release the peptide into their environment, and it was observed that the former released approximately 4% more than the latter over time. This difference in release profile between the two materials is similar to that obtained with the same materials loaded with L-tryptophan, the C-terminal amino acid of PTHrP (107-111)29. It should be taken into account that the negative charges of SBA-15 at the physiological pH used in the process of capture of the peptide promote electrostatic interactions with the slightly positively charge of the amino groups in the PTHrP (107-111) at this pH. However, the hydrophobic surface of C8-SBA-15 is capable of interacting with the indole ring of the tryptophan in this peptide, although this functionalisation leaves a smaller available space (occupied by alkyl rings) for the adsorption of the peptide in the interior of the pores in comparison with SBA-15 29. This may explain the fact that this material retains less PTHrP (107-111) than the C8-SBA-15.
Considering these in vitro findings, we decided to determine the osteogenic capacity of osteostatin loaded in these mesoporous materials in vivo. To this end, we developed a bone regeneration model by causing a cavity defect in rabbit femur. At 4 weeks from the defect, both the SBA-15 and the C8-SBA-15 promoted osteointegration, with the presence of connective and osteoid tissue, in accordance with earlier studies with other ceramic materials35. This effect was greater in the case of both materials with the adsorbed osteostatin, as the presence of neoformed trabeculae in the environs of the biomaterial and a lower thickness of the fibrous capsule indicated, above all after 8 weeks. The lack of inflammatory response with these biomaterials, as the significant absence of cells stained for RAM11 (macrophages) and TRAP indicated, as a consequence of their stability (without apparent degradation), constitutes an advantage of these types of biomaterial36. However, a sustained inflammation associated with the degradation of materials such as β-TCP (ultraporous tricalcium phosphate) or DCaS (dense calcium sulphate) may compromise bone regeneration in bone defects as a consequence of an decoupling of bone regeneration and resorption36. In addition, although the immunostaining for VEGF was faint, it was possible to observe a revascularisation in the defect zone. In this vein, two recent studies by our group have demonstrated that the systemic administration of PTHrP (107-111) stimulates angiogenesis and the VEGF system in the tibia in regeneration in osteopenic diabetic mice, or in those treated with glucocorticoids18,19. The properties of PTH and the N-terminal fragment of PTHrP as anabolic agents when administered systemically in humans and in animal models with osteoporosis and/or bone fracture are known37. Specifically, studies in which PTHrP (1-36) or a synthetic analogue of PTHrP (RS-66271) were used demonstrated an increase in bone mineral density (BMD) in the vertebral spine in postmenopausal women38 as well as in cortical and trabecular bone in osteopenic rats39. Also, RS-66271 administered systemically has been shown to be efficacious in increasing bone repair in rabbits treated with glucocorticoids (osteopenic), increasing BMD and biomechanical parameters and normalising those histological changes associated with bone loss23. On the other hand, different local bone factors, such as IL-1/6, IGF-1 (growth factor similar to type 1 insulin), TGF-β (transforming growth factor) or the bone morphogenetic proteins (BMPs) have been proposed as possible agents for the promotion of bone repair40, although there are few studies of the effects of these factors incorporated in to a material in this context.
The local release of growth factors in bone defects, as represented by the model used in this work, has been demonstrated to be efficacious and advantageous compared with their exogenous administration. Thus, a study in rats with a segmentary defect in the femur and implanted with degradable matrices containing plasmid DNA of BMP4 and/or the fragment 1-34 of PTH increased (to the greatest extent when the matrix contained both factors) bone neoformation in this defect41. Some authors have studied the interaction between endogenous PTHrP and IGF-1 as regulators for bone repair following a fracture in rats42. In this model, in initiating the formation of cartilaginous callus IGF-1 appears to increase chondrogenesis, while PTHrP would regulate the rate of differentiation of chondrocytes and, following endochondrial ossification, the two factors appear to act in a coordinated way to increase osteogenesis through autocrine/paracrine actions. PTHrP, whose expression increases in the preosteoblast cells by means of its N-terminal fragment, could stimulate the differentiation and synthesis of collagen in these cells by means of IGF142. The results obtained in this cavity defect model support the hypothesis that the C-terminal fragment of PTHrP exerts local anabolic effects to promote bone repair, possibly independently of IGF-1, this being based on previous in vitro data13. The data exposed highlight the importance of osteostatin as an anabolic factor released locally in the environs of the implanted materials, as the histology and the µCT analysis show.
The existence of a certain variability in the results obtained may be due to the following reasons: it was attempted to reduce the number of rabbits used in accordance with the ethics guide for the protection of animals, carrying out multi-interventions in both femurs. Also, the reproducibility of the cavity defect, as well as the histological analysis (difficult to cut in paraffin with the material) and of the µCT, had their limitations due to what has already been described above.
In view of the results given, PTHrP (107-111) loaded into the C8-SBA-15 material exerts a higher osteogenic effect in comparison with the non-functionalised material. It is possible that this greater effect is due to the peptide remaining adsorbed in the ceramic, whose residual concentrations have been shown to be sufficient to stimulate the proliferation of the osteoblast cells in vitro, in comparison with SBA-15 in which the peptide is released more rapidly14.

Conclusions
In conclusion, the capacity of the ceramic biomaterials SBA-15 and C8-SBA-15 to support the local release of PTHrP (107-111) favouring the regeneration of bone following a cavity defect in the femur of rabbit is proven. In addition, the functionalisation of SBA-15 with C8 groups and their subsequent loading with PTHrP (107-111) allows the obtaining of an ideal material for the promotion of bone regeneration in this way. The findings here presented support the possible utilisation of these materials loaded with osteostatin as an alternative therapy for the repair and regeneration of bone.

Acknowledgements: This study was carried out thanks to assistance from the Carlos III Institute of Health (Instituto de Salud Carlos III) (PI050117, PI080922, and RETICEF RD06/0013/1002), the Spanish Ministry of Education and Health (Ministerio de Educación y Ciencia de España) (SAF2005-05254), Médica Mutua Madrileña Research Foundation (Fundación de Investigación Médica Mutua Madrileña),the Interministerial Committee for Science and Technology (Comisión Interministerial de Ciencia y Tecnología) (CICYT, Spain) (MAT2008-736) and the Autonomous Community of Madrid (Comunidad Autónoma de Madrid) (S2009/MAT-1472). DL is a post-doctoral researcher associated with the project of the Community of Madrid (Comunidad de Madrid) (S2009/MAT-1472).

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