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The osteoinductive properties of mesoporous silicate coated with osteostatin in a rabbit femur cavity defect model
Biomaterials 31 (2010) 8564e8573
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
The osteoinductive properties of mesoporous silicate coated with osteostatin in a rabbit femur cavity defect model Cynthia G. Trejo a,1, Daniel Lozano b,1, Miguel Manzano c, d, Juan C. Doadrio c, Antonio J. Salinas c, d, Sonia Dapía e, Enrique Gómez-Barrena f, María Vallet-Regí c, d, *, Natalio García-Honduvilla a, Julia Buján a, 2, Pedro Esbrit b, 2 a
Departamento de Especialidades Médicas, Facultad de Medicina, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain Laboratorio de Metabolismo Mineral y Óseo, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (Capio Group), 28040 Madrid, Spain Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain d Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain e Trabeculae Empresa de Base Tecnológica, S.L., 32900, San Cibrao das Viñas, Orense, Spain f Departamento de Traumatología, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (Capio Group), 28040 Madrid, Spain b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2010 Accepted 28 July 2010 Available online 19 August 2010
Parathyroid hormone-related protein (PTHrP) is an important regulator of bone formation and remodeling. Our recent findings demonstrate that PTHrP (107e111) (osteostatin) loaded onto silica-based ordered mesoporous SBA15 materials exhibit osteogenic features in osteoblastic cell cultures. We aimed here to elucidate whether these peptide-coated materials might be suitable for promoting bone repair following a cavitary defect in the rabbit femur. Histological examination revealed the absence of significant inflammation or bone resorption within the time of study (4 and 8 weeks) after implantation. At 8 weeks, the peptide-unloaded materials were still separated from the bone marrow by a fibrous cap, which was greatly diminished by the presence of the PTHrP peptide. By using mCT analysis, new bone formation was evident at different distances from the implants, mainly for the latter peptide-loaded biomaterials. This was confirmed by performing immunostaining for different osteoblast markers. Our findings demonstrate that these PTHrP (107e111)-loaded bioceramics significantly improve local bone induction, as compared to that observed with the unloaded material. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Biocompatibility Bone regeneration Bone tissue engineering Inducing osteogenesis Osteostatine SBA15 mesoporous silica
1. Introduction Parathyroid hormone (PTH)-related protein (PTHrP) is an important modulator of both bone development and bone remodeling [1e3]. Intermittent administration of PTHrP, through its N-terminal domain which presents structural homology to that of PTH, exerts an anabolic action in bone by interaction with the PTH receptor 1 (PTHR1) in osteoblasts [3e7]. In addition, the putative C-terminal PTHrP (107e139) fragment which is unrelated to PTH has shown to exhibit interesting features in bone cells. Thus, it appears to inhibit osteoclastic bone resorption by acting on osteoclastic growth and/or differentiation, apparently through its highly conserved N-terminal sequence 107e111 (osteostatin) * Corresponding author. Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain. Tel.: þ34 91 394 1843; fax: þ34 91 394 1786. E-mail address: [email protected] (M. Vallet-Regí). 1 These authors contributed equally to this work. 2 These authors have the same senior status in this work. 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.07.103
[8e12]. Of interest, PTHrP (107e139) is a substrate for the product of Phex gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) ea metallopeptidase of the M13 family which is present in osteoblasts [13] e, resulting in the production of osteostatin [14]. In addition, several in vitro reports indicate that osteostatin is mitogenic and increases cell differentiation in both rodent and human osteoblastic cells [15e17]. The putative receptor as well as the underlying mechanisms responsible for these effects of PTHrP (107e111) in bone cells are still illdefined [8,18,19]. Insterestingly, our recent report demonstrates that the native fragment of PTHrP containing the sequence 107e139 rapidly transactivates vascular endothelial growth factor (VEGF) receptor 2 in osteoblastic cells [20]. In any event, the true role of this C-terminal PTHrP domain in bone is unclear. An early study showed that daily administration of osteostatin for 2 weeks to ovariectomized rats prevented bone loss in femur and tibia, apparently at the expense of a positive effect in cortical bone [4]. Very recently, we found that an equivalent dose of PTHrP (107e139) than that used for osteostatin in the latter study
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reversed the cortical bone loss and improved bone regeneration after marrow ablation in mice undergoing 3-methylprednisolone administration [21]. These studies suggest an osteogenic effect of this PTHrP domain in vivo, at least in cortical bone, which might be related or not to its anti-resorptive action. New bone formation occurs following injuries such as fractures. In adults, bone repair recapitulates the process of bone formation during development. Thus, the formation of a callus during fracture healing involves both intramembranous and endochondral ossification, but ein contrast to tissue developmente inflammation also occurs [22,23]. In fact, the latter can induce vascularization and thus promotes mesenchymal stem cell growth within the injured bone site. Although the roles of different molecular pathways and cell interactions implicated in the bone healing process are not yet well understood, several strategies are now envisioned as putative therapies to improve this process [23]. One of them involves the local or systemic administration of osteogenic factors that would contribute to increase new bone formation in this scenario. In this regard, previous studies have shown that daily injection of PTH, at doses reported to be anabolic in rodent bone, significantly improved fracture healing in both intact and ovariectomized rats [24,25]. Also of interest, systemic administration of a synthetic PTHrP (1-34) analog (RS-66271; Roche Bioscience, Palo Alto, CA) was shown to counteract the prednisone-induced impairment of an ulna defect healing in rabbits [26]. A more direct approach to stimulate bone repair using this rationale, however, might be the local delivery of osteogenic factors into the injured bone site. To achieve this, silica-based ordered mesoporous matrices (SBA15) have been largely employed as drug delivery systems [27e29]. We recently characterized the uptake and release kinetics of PTHrP (107e111) loaded into these matrices with different degree of hydrophobicity on their silica surface. Our in vitro findings demonstrate that this peptide confers osteogenic activity to both unmodified and organically-modified SBA15 [17]. The aim of the present study was to evaluate whether the aforementioned PTHrP (107e111)-loaded biomaterials might retain their osteogenic capacity in vivo. Therefore, we evaluated the relative osteointegration of both functionalized and non-functionalized SBA15 ceramics, loaded or not with this peptide, and their influence on surrounding bone when implanted into a cavitary defect in the rabbit femur. 2. Materials and methods 2.1. Preparation of materials SBA15 was synthesized using a surfactant (Pluronic P123, BASF, New Jersey, USA) as structure-directing agent and tetraethylorthosilicate (SigmaeAldrich, St Louis, MO) as silica source, as described elsewhere [30]. Its ordered mesoporous structure was confirmed by small angle X-ray diffraction and N2 adsorption analysis. An organic modification of the silica walls was carried out by grafting an alkoxysilane, n-octyltriethoxysilane (C8, SigmaeAldrich), as described [17]. 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 0.05 g disks (6 2 mm) by uniaxial (1 MPa) and isostatic pressure (1 MPa). Loading with human PTHrP (107e111) 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 [17]. Materials were exposed to UV irradiation for esterilization prior to surgery. 2.2. Surgical procedures Our protocol, using a limited number of female New Zealand rabbits (6 months of age; n ¼ 4 per experimental group) by means of multisite intervention, was approved by the Institutional Animal Care and Use Committee at the Instituto de Investigación Sanitaria-Fundación Jiménez Díaz, according to the European Union guidelines to decrease pain and suffering of the animals. Surgical intervention was performed under general anesthesia. Lateral and medial approaches were performed in both shaved knees to exposure the distal femoral epiphysis. Lateral and medial cavitary defects were created with a medium speed burr (5 mm diameter
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and 4e5 mm depth). Tested materials were then implanted and the wounds were sutured. Lateral and medial right femoral defects received unloaded and PTHrP (107e111)-loaded SBA15 materials, respectively. Meanwhile, lateral and medial left femoral defects received unloaded and PTHrP (107e111)-loaded SBA15-C8 materials, respectively. At 4 and 8 weeks after implantation, femora were removed and assigned to histological and immunohistochemical studies and micro-computerized tomography (mCT) analysis. 2.3. Histological and immunohistochemical studies Femora removed were fixed in 10% neutral formaldehyde, decalcified with Osteosoft (Merck, Madrid, Spain), and then dehydrated before paraffin embedding. All histological and inmunohistochemical determinations were carried out onto sagittal 5e8-mm sections/sample in a Zeiss Axiophot optical microscope (Carl Zeiss, Oberkochen, Germany). For histological analysis, hematoxylin/eosin and Masson’s trichromic staining were used. For immunohistochemistry, deparaffinized and rehydrated tissue sections were first incubated in 4% bovine serum albumin in PBS containing 0.1% Triton X-100 for 30 min at room temperature. The following primary mouse monoclonal antibodies were used (dilution, fold): anti-osteopontin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (50); anti-osteocalcin antibody (Santa Cruz Biotechnology) (50); anti-Runx2 antibody (Santa Cruz Biotechnology) (50); tartrate-resistant acid phosphatase (TRAP) antibody (Santa Cruz Biotechnology) (100); proliferating cell nuclear antigen (PCNA) (ABCAM, Cambridge, UK) (50); monocyte/macrophage antibody (RAM11; Dako, Glostrup, Denmark) (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, except for sclerostin which were incubated for 2 h at room temperature. Sections were subsequently incubated with biotinlylated anti-goat IgG and avidinebiotine horse radish peroxidase (HRP) complex (Dako) or HRPerabbite anti-goat IgG (sclerostin), followed by 3,30 -diaminobenzidine as chromogen. Sections were counterstained with hematoxylin. The number of immunostained cells for every parameter studied were counted in 10 consecutive x400-fields in the same area in the vicinity of the implanted materials. For sclerostin, this area was restricted to that containing cortical bone. The results were expressed as percentage of positive cells by respect to total (showing positive and negative staining) cells in the evaluated area. All stainings were evaluated by 2e3 independent observers in a blinded fashion, and the corresponding mean score value was obtained for each animal. 2.4. mCT analysis Femora from 4 rabbits in each group were cut with a diamond disk (Komet Group, Lemgo, Germany) and then scanned with a high-resolution microtomographic system (SkyScan 1172, Skyscan N.V., Aartselaar, Belgium). Samples were imaged at a scanning voxel size of 10.9 mm, X-ray tube voltage of 100 kV and current of 100 mA, without filter. The scanning angular rotation was 360 , and the angular increment was 0.40 . Images were reconstructed based on Feldkamp convolution backprojection algorithm, and segmented into binary images (8-bit BMP images) using adaptive local thresholding. For determination of the 3-D microarchitectural properties within the bone regeneration area, specimens were evaluated using standard software (SkyScanÔ CT-analyzer software, version 1.7.0.5). The % bone volume (BV/ TV) and trabecular bone pattern factor (Tb.Pf) were calculated at different distances from the biomaterial surface (at 5, 10, 15 and 20 pixels; 1 pixel z21.8 mm). 2.5. Statistical analysis Results are expressed as mean SD. Statistical analysis was performed by non parametric t-test with statistical software GraphPad Prism 5.0 for Windows XP. p < 0.05 was considered significant.
3. Results 3.1. Histological evaluation For evaluating the tissue response to the biomaterial and the repair process, we performed mCT and histological analysis on the tissue/biomaterial interface and the peripheral area of the implant. By using the former analysis, new bone formation was evident at different distances from the implant, which was more dramatic for the osteostatin-loaded biomaterials at both time periods studied (Figs. 1 and 2, upper panels). The osteoinductive effect of osteostatin was confirmed by quantification of % BV/TV and Pb.Tf. in these bone samples (Table 1). After 4 weeks of implantation, the unloaded materials were intact within the cavitary defect, surrounded by a fibrous cap ewhich
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Fig. 1. Tridimensional reconstruction images and saggital images by mCT (upper panels) and light microscopy (from hematoxylin/eosin stained sections) (lower panels) of the area surrounding the different implants (m), containing newly formed bone at different distances from each implanted material at 4 weeks after production of a cavitary defect in the rabbit femur.
was thicker in the case of SBA15-C8e in close proximity to the material, and abundant connective tissue formed the cortical surface of the callus (Fig. 1). Four weeks later, the materials were still separated from the bone marrow by the fibrous cap, but newly formed small trabeculae were evident in the area of the callus close
to the implant, which were more abundant in the vicinity of SBA15 (Fig. 2). Implantation of both osteostatin-loaded SBA15 and SBA15-C8 materials promoted tissue healing after 4 weeks, as shown by the appearance of abundant osteoid and new trabeculae all around the
Fig. 2. Tridimensional reconstruction images by mCT (upper panels) and light microscopy (from hematoxylin/eosin stained sections) (lower panels) of the area surrounding the different implants, containing newly formed bone at different distances from each implanted material at 8 weeks after production of a cavitary defect in the rabbit femur.
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Table 1 Bone structural parameters at different distances from the different implants tested at 4 and 8 weeks following production of a cavitary defect in the rabbit femur. SBA15 þ PTHrP (107e111)
4 weeks
SBA15
Distance (mm)
BV/TV (%)
Tb.Pf (mm1)
BV/TV (%)
Tb.Pf (mm1)
BV/TV (%)
Tb.Pf (mm1)
BV/TV (%)
Tb.Pf (mm1)
218 436
0.97 10.43
40.23 16.43
10.43 25.71
8.53 5.02
2.32 8.68
35.30 18.95
20.50 18.15
4.95 9.53
SBA15 þ PTHrP (107e111)
SBA15-C8
SBA15-C8þPTHrP (107e111)
8 weeks
SBA15
Distance (mm)
BV/TV (%)
Tb.Pf (mme1)
BV/TV (%)
Tb.Pf (mme1)
BV/TV (%)
SBA15-C8 Tb.Pf (mme1)
BV/TV (%)
SBA15-C8þPTHrP (107e111) Tb.Pf (mme1)
218 436
19.91 33.60
16.88 9.59
33.23 46.45
9.38 7.12
1.17 1.14
38.23 34.05
6.40 34.84
10.03 2.90
BV/TV, bone volume/total volume; Tb.Pf., bone trabecular pattern factor. Data correspond to a representative rabbit in each experimental group.
biomaterial (Fig. 1, lower panel). After 8 weeks, newly formed bone was predominant in the repairing area, as trabecular and cortical structures, and a hypertrophic scar appears toward the exterior of the implant; meanwhile, trabeculae were observed around the biomaterial in the vicinity of bone marrow (Fig. 2, lower panel). Moreover, at this time period, the fibrous cap was split, allowing osteoid tissue to be in contact with the biomaterial, in the osteostatin-loaded SBA15-C8 group (Fig. 2, lower panel). 3.2. Inmunohistochemistry RAM11, a specific antibody against rabbit monocytes/macrophages, was used to confirm the biocompatibility (no inflammation induction) of each tested material (Fig. 3). A low number of RAM11positive cells were detected in the vicinity of the implanted materials during the time of study. Macrophages (as RAM11-positive cells) appeared at the each material/tissue interface at 4 weeks post-implant. At 8 weeks, however, this response decreased in the SBA15-C8 group, related to fibrous cap formation (Fig. 3A and B). RAM11-positive macrophages in a row are clearly displayed at the
SBA15-C8/tissue interface at 4 weeks (Fig. 3C). These cells were still detected in the outer layer of the fibrous cap surrounding the implanted SBA15 material (Fig. 3D). Of interest, the presence of PTHrP (107e111) was found to dramatically decrease this macrophage infiltration associated to both types of tested biomaterials at the latter time period (Fig. 3A and B). A high proliferative activity (as PCNA positivity) was found in the wounded soft tissue, both in connective tissue and the vascular bed. After 4 weeks, the scar process maintains a high proliferative activity in all groups studied; so that the mean score values for PCNA positivity were: 10.7% and 6.6% for the same area around SBA15 and SBA15-C8 materials, respectively. Meanwhile, these values were: 17.6% and 20.9% for the corresponding areas close to each PTHrP (107e111)-loaded biomaterials, respectively. At 8 weeks, this proliferation rate dramatically decreased in all the specimens studied, so that PCNA staining remained detectable (<10% of cells/field) only in the case of peptide-loaded SBA15-C8 (Fig. 4). Nuclear immunostaining for the transcription factor Runx2 was detected in cells around both tested materials, SBA15 and SBA15-
Fig. 3. Changes in RAM11 immunostaining in the area surrounding the implanted materials during bone regeneration. Comparative score values at 4 weeks (A) and 8 weeks (B). *p < 0.001, SBA15-C8 þ PTHrP (107e111) vs. SBA15 þ PTHrP (107e111); ap<0.001, SBA15-C8 vs. SBA15-C8 þ PTHrP (107e111); bp < 0.05, SBA15 vs. SBA15 þ PTHrP (107e111); and c p < 0.05 SBA15 vs. SBA15-C8. Images show the presence of RAM11-positive macrophages (denoted by arrows) in the fibrous cap (FC) close to the implanted material (m) in representative samples from the SBA15-C8 group at 4 weeks (C) or from the SBA15 group at 8 weeks (D).
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Fig. 4. Representative PCNA immunostaining showing labeled cells (arrows) at the interface between material (m) (SBA15-C8 þ PTHrP (107e111)) and fibrous cap (FC) at 4 weeks (A) or in the split fibrous cap at 8 weeks (B) within the femoral defect in rabbit.
C8. It was similar in all the groups studied after 4 weeks during the repair process (Fig. 5A); whereas PTHrP (107e111) loading onto these materials, mainly in the case of SBA15-C8, significantly increased the number of Runx2-positive cells without affecting the cell staining pattern after 8 weeks (Fig. 5B). Abundant nuclear staining for Runx2 was evident in newly formed bone areas around PTHrP (107e111)-loaded SBA15-C8 biomaterial, compared to that observed close to peptide-unloaded SBA15-C8 material, at 8 weeks (Fig. 5C and D).
After 4 weeks, the number of osteopontin-positive cells was higher for PTHrP (107e111)-loaded SBA15-C8 biomaterial than for the other groups tested during the period of study (Fig. 6A). This positivity increased near SBA15, independent of the presence of the PTHrP peptide, at the end of study (8 weeks) (Fig. 6A and B). Similar changes were also observed in immunostaining score values for osteocalcin ea late osteoblast differentiation marker that is related to the mineralization processe in the bone healing tissue (Fig. 7AeC). Thus, at 4 and 8 weeks after implantation, these
Fig. 5. Runx2 immunostaining in the implant bone interface within the femoral defect in rabbits. Comparative score values at 4 weeks (A) and 8 weeks (B) bp < 0.001, SBA15 vs. SBA15 þ PTHrP (107e111); ap < 0.001, SBA15-C8 vs. SBA15-C8 þ PTHrP (107e111). Images show abundant positivity (arrows) for this transcription factor in cells around newly formed bone areas corresponding to SBA15-C8 þ PTHrP (107e111) (C), which contrasts to the poor staining in these areas close to this peptide-unloaded material (D), at 8 weeks. Blood vessels were evident in these microimages.
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Fig. 6. Changes in osteopontin immunostaining in the implant bone interface within the femoral defect in rabbits. Comparative score values at 4 weeks (A) *p < 0,05 SBA15 þ PTHrP (107e111) vs. SBA15-C8 þ PTHrP (107e111) and ap < 0,01 SBA15-C8 vs. SBA15-C8 þ PTHrP (107e111). At 8 weeks (B) ap < 0.001, C8-SBA15 vs. C8-SBA15 þ PTHrP (107e111). A representative image showing the pattern of osteopontin staining (denoted by arrows) corresponding to SBA15 þ PTHrP (107e111) at 8 weeks is shown (C).
staining values were significantly higher in this tissue around PTHrP (107e111)-loaded SBA15-C8 biomaterial than in the other groups studied. Interestingly, at 8 weeks, staining for this marker increased in both SBA15 groups tested and also in the PTHrP (107e111)-loaded SBA15-C8 group but not in the group containing the latter material alone (Fig. 7C). Considering the well characterized relationship between angiogenesis and osteogenesis [31], and the previously reported effect of osteostatin and/or the native C-terminal PTHrP fragment 107e139 on induction of the VEGF system in bone [16,20,21], we sought to assess VEGF immunostaining in the bone regenerating tissue close to the implanted materials. We found VEGF-positive cells in the healing tissue, including blood vessels and mesenchymal cells associated with high osteoid formation, only in the case of the PTHrP (107e111)-loaded biomaterials at 8 weeks (Fig. 8A and B). Sclerostin, the product of SOST gene that is specifically expressed in osteocytes, acts as an endogenous modulator of the canonical Wnt pathway and thus of bone formation [32,33]. Positivity for this marker in the repairing area showed a significant relationship to the presence of PTHrP (107e111) during the period of study (Fig. 9A and B). After 8 weeks, we observed a significant decrease for this positivity in osteocytes within this area close to the peptide-loaded SBA15-C8 biomaterial to values similar to those for the corresponding non-functionalized biomaterial (Fig. 9C). This might indicate that stabilization of the osteogenic process has occurred at this time with each type of tested biomaterial. To further support the hypothesis that this might be the case, we evaluated the presence of osteoclasts using
a specific TRAP anti-antibody. We found very few TRAP-positive polynucleated cells (osteoclasts) in all of the rabbit tissue samples studied; this was specially the case in the vicinity of SBA15-C8, mainly when PTHrP (107e111) was present (Fig. 10). Osteoclasts are cells derived from the monocyte/macrophage lineage. In fact, RAM11-positive cells were more abundant than TRAP-positive osteoclasts at 4 weeks, and similar to what was observed for the latter cells, the former cells dramatically decreased by the presence of the PTHrP peptide onto the implant (Figs. 3 and 10). 4. Discussion In our recent in vitro report [17], we showed that osteostatinloaded SBA15-based biomaterials display osteogenic features in osteoblastic cells. In this previous study, we found that osteostatin was released as a burst from both SBA15 and SBA15-C8 materials within the first 24 h in cell culture medium. However, even osteostatin remaining in these biomaterials and released at concentrations in the < nM range during prolonged incubations (up to 12 days) was efficient to affect osteoblastic function in vitro [17]. In this study, we also showed the higher efficacy of osteostatinloaded SBA15-C8 than that of its SBA15 counterpart to modulate the expression of some osteoblastic genes, namely osteocalcin (OC) and osteoprotegerin (OPG) [17]; likely as a consequence of the greater peptide adsorption to the external surface of the former functionalized biomaterial than in the case of SBA15, making it more accessible to the osteoblastic cell microenvironment. These in
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Fig. 7. Changes in osteocalcin staining in the area surrounding the different implants during bone regeneration within a femoral defect. Comparative score values at both time periods studied, 4 (A) and 8 weeks (B). *p < 0.02, C8eSBA15 þ PTHrP (107e111) vs. SBA15 þ PTHrP (107e111); ap < 0.001, C8-SBA15 vs. SBA15-C8 þ PTHrP (107e111); bp < 0.05, SBA15 vs. SBA15 þ PTHrP (107e111). #p < 0.01, between 4 and 8 weeks (C) for SBA15, SBA15 þ PTHrP (107e111) and SBA15-C8 þ PTHrP (107e111) implants. Image shows the presence of osteocalcinepositive cells in a representative tissue sample from the SBA15-C8 þ PTHrP (107e111) group at 8 weeks (D). Arrows denote the presence of osteocalcinpositive cells (osteoblasts).
vitro findings demonstrate the osteogenic features conferred by osteostatin coating onto SBA15-based materials, which prompted us to test them as putative biomaterials to promote bone regeneration in vivo. In the present study, we assessed the in vivo effects of PTHrP (107e111) delivered by SBA15 materials into a cavitary bone defect located in the epiphysis of the rabbit femur. This is an environment made up of trabecular bone and a minor cortical bone component
at the edge of the defect, thus unexposed to mechanical strains, which has been previously proven suitable for testing biomaterials [34,35]. Using this animal model, we have been able to show that these peptide-loaded bioceramics exert a clear bone regeneration action (as assessed by histological and mCT analysis and immunohistochemistry). Furthermore, our findings herein demonstrate that these PTHrP (107e111)-loaded biomaterials are highly osteoconductive and
Fig. 8. Representative image showing VEGF positivity in blood vessels (*) in regenerating bone (RB) (A) and in stromal cells within the bone marrow (arrows) close to the SBA15C8 þ PTHrP (107e111) biomaterial (m) (B) at 8 weeks. FC ¼ fibrous cap.
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Fig. 9. Changes in sclerostin immunostaining in the area surrounding the different implants in a femoral defect in rabbits. Comparative score values at 4 (A) and 8 weeks (B). a p < 0.001, C8-SBA15 vs. SBA15-C8þPTHrP (107e111); bp < 0.05, SBA15 vs. SBA15 þ PTHrP (107e111); #p < 0.02, between 4 and 8 weeks (C) for SBA15-C8þPTHrP (107e111). Image shows the presence of sclerotin-positive cells (osteocytes; arrows) in regenerating bone in a representative sample from the SBA15 þ PTHrP (107e111) group at 8 weeks (D).
osteoinductive by promoting infiltration of proliferating osteogenic precursor cells and connective tissue formation. In fact, the appearance of a thick fibrous cup induced by SBA15-C8 was significantly reduced by PTHrP (107e111) coating, associated with increased bone regeneration. The latter was further assessed by immunostaining for various osteoblast markers in the tissue
surrounding the implant. During the initial phase of the repair process (4 weeks), Runx2-positive cells increased in the newly formed trabeculae infiltrating the defect independent of the type of implant tested. This positivity decreased with time after implantation of both types of ceramics but remained high in the presence of PTHrP (107e111), related to an increased osteopontin and OC
Fig. 10. TRAP immunostaining in the healing bone area close to each implant after a femoral defect in rabbit. Comparative score values at 4 weeks (A) and 8 weeks (B). *p < 0.05, C8-SBA15 þ PTHrP (107e111) vs. SBA15 þ PTHrP (107e111); ap < 0.05, SBA15-C8 vs. SBA15-C8þPTHrP (107e111); cp < 0.02, SBA15 vs. SBA15-C8. Representative image showing the presence of TRAP-positive cells (arrows) in the SBA15-C8 group at 4 weeks.
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staining; the latter was specially so in the case of SBA15-C8 material. This is totally consistent with the crucial role of Runx2 for modulation of the latter osteoblastic differentiation genes [36]. In fact, this transcription factor has recently been shown to be a target of low-intensity ultrasounds to improve osteoblastic cell differentiation in delayed unions in humans [37]. This osteogenic effect of PTHrP (107e111) which extended the maturation phase during regeneration was associated with an increased cell proliferation (based on PCNA immunostaining) and an augmented number of sclerostin-positive osteocytes in cortical bone close to the implant. In addition, the PTHrP (107e111)-loaded biomaterials induced revascularization of the defect, related to an increased immunostaining for VEGF in the healing bone tissue. This is particularly interesting since systemic administration of PTHrP (107e139) has recently been shown to stimulate angiogenesis and the VEGF system in the regenerating tibia of mice with glucocorticoidinduced osteopenia [21]. In fact, current data underscore the key role of the latter system on the process of neoangiogenesis and new bone formation in the setting of bone healing [31,38]. Some monocytes/macrophages (based on RAM11 positivity) infiltrated the regenerating tissue around the implants during the period of study. For the functionalized SBA15 material, this moderate inflammatory response was transient, since RAM11 positivity was absent at 8 weeks post-implantation. Moreover, PTHrP (107e111) loading dramatically inhibited this response as well as the appearance of TRAP-positive osteoclasts in the vicinity of the implant. The latter was expected in view of the previously reported inhibitory effect of PTHrP (107e111) on osteoclastogenesis in vitro [8,9], and that of PTHrP (107e139) in vivo in the regenerating tibia of glucocorticoid-treated mice [21]. The present findings further support the notion that this PTHrP domain has anti-resorptive features, and suggest that PTHrP (107e111) inhibits monocyte/macrophage differentiation towards the osteoclastic lineage during bone regeneration in this bone healing model. 5. Conclusions The present report demonstrates that PTHrP (107e111)-coated bioceramics are efficient in favoring peri-implant bone regeneration in an experimental cavitary defect, as compared with the bioceramic alone. Moreover, SBA15 functionalization with C8 followed by PTHrP (107e111) coating allowed us to obtain a suitable biomaterial for local delivery of the peptide to promote bone regeneration in this setting. These findings could make these biomaterials as potential formulations for clinical application. Acknowledgments This study was supported by grants from Instituto de Salud Carlos III(PI050117, PI080922, and RETICEFRD06/0013/1002 and RD06/0013/1006), Ministerio de Educación y Ciencia (SAF2005e05254), Fundación de Investigación Médica Mutua Madrileña, Comisión Interministerial de Ciencia y Tecnología (CICYT, Spain)(MAT2008e736) and Comunidad Autónoma de Madrid (S2009/MATe1472). DL was supported by Fundación Conchita Rábago. Appendix Figures with essential color discrimination. Figs. 1e10 in this article have parts that are difficult to interpret in black and white. The full color images can be found in the online version, at doi:10. 1016/j.biomaterials.2010.07.103.
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Osteostatin-loaded bioceramics stimulate osteoblastic growth and differentiation. Acta Biomater 2010;6:797e803. [18] Valín A, Guillén C, Esbrit P. C-terminal parathyroid hormone-related protein (PTHrP) (107e139) stimulates intracellular Ca2þ through a receptor different from the type 1 PTH/PTHrP receptor in osteoblastic osteosarcoma UMR 106 cells. Endocrinology 2001;142:2752e9. [19] Guillén C, Martínez P, de Gortázar AR, Martínez ME, Esbrit P. Both N- and C-terminal domains of parathyroid hormone-related protein increase interleukin-6 by NF-kB activation in osteoblastic cells. J Biol Chem 2002;277: 28109e17. [20] Alonso V, de Gortázar AR, Ardura JA, Andrade-Zapata I, Álvarez-Arroyo MV, Esbrit P. Parathyroid hormone-related protein (107e139) increases human osteoblastic cell survival by activation of vascular endothelial growth factor receptor-2. J Cell Physiol 2008;217:717e27. [21] de Castro LF, Lozano D, Dapía S, PortaleNúñez S, Caeiro JR, GómezeBarrena E, et al. Role of the N- and C-terminal fragments of parathyroid hormone-related protein as putative therapies to improve bone regeneration under high glucocorticoid treatment. Tissue Eng Part A 2010;16:1157e68. [22] Ferguson CM, Miclau T, Hu D, Alpern E, Helms JA. Common molecular pathways in skeletal morphogenesis and repair. Ann NY Acad Sci 1998;857: 33e42. [23] Deschaseaux F, Sensébé L, Heymann D. Mechanisms of bone repair and regeneration. Trends Mol Med 2009;15:417e29. [24] Walsh WR, Sherman P, Howlett CR, Sonnabend DH, Ehrlich MG. Fracture healing in a rat osteopenia model. Clin Orthop Relat Res 1997;342:218e27.
C.G. Trejo et al. / Biomaterials 31 (2010) 8564e8573 [25] Andreassen TT, Ejersted C, Oxlund H. Intermittent parathyroid hormone (1e34) treatment increases callus formation and mechanical strength of healing rat fractures. J Bone Miner Res 1999;14:960e8. [26] Bostrom MP, Gamradt SC, Asnis P, Vickery BH, Hill E, Avnur Z, et al. Parathyroid hormone-related protein analog RS-66271 is an effective therapy for impaired bone healing in rabbits on corticosteroid therapy. Bone 2000;26:437e42. [27] ValleteRegí M, Balas F, Arcos D. Mesoporous materials for drug delivery. Angew Chem Int Ed 2007;46:7548e58. [28] ValleteRegi M, Balas F, Colilla M, Manzano M. Bone-regenerative bioceramic implants with drug and protein controlled delivery capability. Prog Solid State Chem 2008;36:163e91. [29] Manzano M, ValleteRegí M. New developments in ordered mesoporous materials for drug delivery. J Mater Chem 2010;20:5593e604. [30] Zhao D, Hou Q, Fena J, Chmelka BF, Stucky GD. Non ionic triblock and star diblock copolymer and oligomeric surfactant synthesis of highly ordered, hydrothermally stable, mesoporous silica structures. J Am Chem Soc 1998;120:6024e36. [31] Schipani E, Maes C, Carmeliet G, Semenza GL. Regulation of osteogenesis-angiogenesis coupling by HIFs and VEGF. J Bone Miner Res 2009;24: 1347e53.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
The osteoinductive properties of mesoporous silicate coated with osteostatin in a rabbit femur cavity defect model Cynthia G. Trejo a,1, Daniel Lozano b,1, Miguel Manzano c, d, Juan C. Doadrio c, Antonio J. Salinas c, d, Sonia Dapía e, Enrique Gómez-Barrena f, María Vallet-Regí c, d, *, Natalio García-Honduvilla a, Julia Buján a, 2, Pedro Esbrit b, 2 a
Departamento de Especialidades Médicas, Facultad de Medicina, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain Laboratorio de Metabolismo Mineral y Óseo, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (Capio Group), 28040 Madrid, Spain Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain d Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain e Trabeculae Empresa de Base Tecnológica, S.L., 32900, San Cibrao das Viñas, Orense, Spain f Departamento de Traumatología, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (Capio Group), 28040 Madrid, Spain b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2010 Accepted 28 July 2010 Available online 19 August 2010
Parathyroid hormone-related protein (PTHrP) is an important regulator of bone formation and remodeling. Our recent findings demonstrate that PTHrP (107e111) (osteostatin) loaded onto silica-based ordered mesoporous SBA15 materials exhibit osteogenic features in osteoblastic cell cultures. We aimed here to elucidate whether these peptide-coated materials might be suitable for promoting bone repair following a cavitary defect in the rabbit femur. Histological examination revealed the absence of significant inflammation or bone resorption within the time of study (4 and 8 weeks) after implantation. At 8 weeks, the peptide-unloaded materials were still separated from the bone marrow by a fibrous cap, which was greatly diminished by the presence of the PTHrP peptide. By using mCT analysis, new bone formation was evident at different distances from the implants, mainly for the latter peptide-loaded biomaterials. This was confirmed by performing immunostaining for different osteoblast markers. Our findings demonstrate that these PTHrP (107e111)-loaded bioceramics significantly improve local bone induction, as compared to that observed with the unloaded material. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Biocompatibility Bone regeneration Bone tissue engineering Inducing osteogenesis Osteostatine SBA15 mesoporous silica
1. Introduction Parathyroid hormone (PTH)-related protein (PTHrP) is an important modulator of both bone development and bone remodeling [1e3]. Intermittent administration of PTHrP, through its N-terminal domain which presents structural homology to that of PTH, exerts an anabolic action in bone by interaction with the PTH receptor 1 (PTHR1) in osteoblasts [3e7]. In addition, the putative C-terminal PTHrP (107e139) fragment which is unrelated to PTH has shown to exhibit interesting features in bone cells. Thus, it appears to inhibit osteoclastic bone resorption by acting on osteoclastic growth and/or differentiation, apparently through its highly conserved N-terminal sequence 107e111 (osteostatin) * Corresponding author. Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain. Tel.: þ34 91 394 1843; fax: þ34 91 394 1786. E-mail address: [email protected] (M. Vallet-Regí). 1 These authors contributed equally to this work. 2 These authors have the same senior status in this work. 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.07.103
[8e12]. Of interest, PTHrP (107e139) is a substrate for the product of Phex gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) ea metallopeptidase of the M13 family which is present in osteoblasts [13] e, resulting in the production of osteostatin [14]. In addition, several in vitro reports indicate that osteostatin is mitogenic and increases cell differentiation in both rodent and human osteoblastic cells [15e17]. The putative receptor as well as the underlying mechanisms responsible for these effects of PTHrP (107e111) in bone cells are still illdefined [8,18,19]. Insterestingly, our recent report demonstrates that the native fragment of PTHrP containing the sequence 107e139 rapidly transactivates vascular endothelial growth factor (VEGF) receptor 2 in osteoblastic cells [20]. In any event, the true role of this C-terminal PTHrP domain in bone is unclear. An early study showed that daily administration of osteostatin for 2 weeks to ovariectomized rats prevented bone loss in femur and tibia, apparently at the expense of a positive effect in cortical bone [4]. Very recently, we found that an equivalent dose of PTHrP (107e139) than that used for osteostatin in the latter study
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reversed the cortical bone loss and improved bone regeneration after marrow ablation in mice undergoing 3-methylprednisolone administration [21]. These studies suggest an osteogenic effect of this PTHrP domain in vivo, at least in cortical bone, which might be related or not to its anti-resorptive action. New bone formation occurs following injuries such as fractures. In adults, bone repair recapitulates the process of bone formation during development. Thus, the formation of a callus during fracture healing involves both intramembranous and endochondral ossification, but ein contrast to tissue developmente inflammation also occurs [22,23]. In fact, the latter can induce vascularization and thus promotes mesenchymal stem cell growth within the injured bone site. Although the roles of different molecular pathways and cell interactions implicated in the bone healing process are not yet well understood, several strategies are now envisioned as putative therapies to improve this process [23]. One of them involves the local or systemic administration of osteogenic factors that would contribute to increase new bone formation in this scenario. In this regard, previous studies have shown that daily injection of PTH, at doses reported to be anabolic in rodent bone, significantly improved fracture healing in both intact and ovariectomized rats [24,25]. Also of interest, systemic administration of a synthetic PTHrP (1-34) analog (RS-66271; Roche Bioscience, Palo Alto, CA) was shown to counteract the prednisone-induced impairment of an ulna defect healing in rabbits [26]. A more direct approach to stimulate bone repair using this rationale, however, might be the local delivery of osteogenic factors into the injured bone site. To achieve this, silica-based ordered mesoporous matrices (SBA15) have been largely employed as drug delivery systems [27e29]. We recently characterized the uptake and release kinetics of PTHrP (107e111) loaded into these matrices with different degree of hydrophobicity on their silica surface. Our in vitro findings demonstrate that this peptide confers osteogenic activity to both unmodified and organically-modified SBA15 [17]. The aim of the present study was to evaluate whether the aforementioned PTHrP (107e111)-loaded biomaterials might retain their osteogenic capacity in vivo. Therefore, we evaluated the relative osteointegration of both functionalized and non-functionalized SBA15 ceramics, loaded or not with this peptide, and their influence on surrounding bone when implanted into a cavitary defect in the rabbit femur. 2. Materials and methods 2.1. Preparation of materials SBA15 was synthesized using a surfactant (Pluronic P123, BASF, New Jersey, USA) as structure-directing agent and tetraethylorthosilicate (SigmaeAldrich, St Louis, MO) as silica source, as described elsewhere [30]. Its ordered mesoporous structure was confirmed by small angle X-ray diffraction and N2 adsorption analysis. An organic modification of the silica walls was carried out by grafting an alkoxysilane, n-octyltriethoxysilane (C8, SigmaeAldrich), as described [17]. 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 0.05 g disks (6 2 mm) by uniaxial (1 MPa) and isostatic pressure (1 MPa). Loading with human PTHrP (107e111) 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 [17]. Materials were exposed to UV irradiation for esterilization prior to surgery. 2.2. Surgical procedures Our protocol, using a limited number of female New Zealand rabbits (6 months of age; n ¼ 4 per experimental group) by means of multisite intervention, was approved by the Institutional Animal Care and Use Committee at the Instituto de Investigación Sanitaria-Fundación Jiménez Díaz, according to the European Union guidelines to decrease pain and suffering of the animals. Surgical intervention was performed under general anesthesia. Lateral and medial approaches were performed in both shaved knees to exposure the distal femoral epiphysis. Lateral and medial cavitary defects were created with a medium speed burr (5 mm diameter
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and 4e5 mm depth). Tested materials were then implanted and the wounds were sutured. Lateral and medial right femoral defects received unloaded and PTHrP (107e111)-loaded SBA15 materials, respectively. Meanwhile, lateral and medial left femoral defects received unloaded and PTHrP (107e111)-loaded SBA15-C8 materials, respectively. At 4 and 8 weeks after implantation, femora were removed and assigned to histological and immunohistochemical studies and micro-computerized tomography (mCT) analysis. 2.3. Histological and immunohistochemical studies Femora removed were fixed in 10% neutral formaldehyde, decalcified with Osteosoft (Merck, Madrid, Spain), and then dehydrated before paraffin embedding. All histological and inmunohistochemical determinations were carried out onto sagittal 5e8-mm sections/sample in a Zeiss Axiophot optical microscope (Carl Zeiss, Oberkochen, Germany). For histological analysis, hematoxylin/eosin and Masson’s trichromic staining were used. For immunohistochemistry, deparaffinized and rehydrated tissue sections were first incubated in 4% bovine serum albumin in PBS containing 0.1% Triton X-100 for 30 min at room temperature. The following primary mouse monoclonal antibodies were used (dilution, fold): anti-osteopontin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (50); anti-osteocalcin antibody (Santa Cruz Biotechnology) (50); anti-Runx2 antibody (Santa Cruz Biotechnology) (50); tartrate-resistant acid phosphatase (TRAP) antibody (Santa Cruz Biotechnology) (100); proliferating cell nuclear antigen (PCNA) (ABCAM, Cambridge, UK) (50); monocyte/macrophage antibody (RAM11; Dako, Glostrup, Denmark) (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, except for sclerostin which were incubated for 2 h at room temperature. Sections were subsequently incubated with biotinlylated anti-goat IgG and avidinebiotine horse radish peroxidase (HRP) complex (Dako) or HRPerabbite anti-goat IgG (sclerostin), followed by 3,30 -diaminobenzidine as chromogen. Sections were counterstained with hematoxylin. The number of immunostained cells for every parameter studied were counted in 10 consecutive x400-fields in the same area in the vicinity of the implanted materials. For sclerostin, this area was restricted to that containing cortical bone. The results were expressed as percentage of positive cells by respect to total (showing positive and negative staining) cells in the evaluated area. All stainings were evaluated by 2e3 independent observers in a blinded fashion, and the corresponding mean score value was obtained for each animal. 2.4. mCT analysis Femora from 4 rabbits in each group were cut with a diamond disk (Komet Group, Lemgo, Germany) and then scanned with a high-resolution microtomographic system (SkyScan 1172, Skyscan N.V., Aartselaar, Belgium). Samples were imaged at a scanning voxel size of 10.9 mm, X-ray tube voltage of 100 kV and current of 100 mA, without filter. The scanning angular rotation was 360 , and the angular increment was 0.40 . Images were reconstructed based on Feldkamp convolution backprojection algorithm, and segmented into binary images (8-bit BMP images) using adaptive local thresholding. For determination of the 3-D microarchitectural properties within the bone regeneration area, specimens were evaluated using standard software (SkyScanÔ CT-analyzer software, version 1.7.0.5). The % bone volume (BV/ TV) and trabecular bone pattern factor (Tb.Pf) were calculated at different distances from the biomaterial surface (at 5, 10, 15 and 20 pixels; 1 pixel z21.8 mm). 2.5. Statistical analysis Results are expressed as mean SD. Statistical analysis was performed by non parametric t-test with statistical software GraphPad Prism 5.0 for Windows XP. p < 0.05 was considered significant.
3. Results 3.1. Histological evaluation For evaluating the tissue response to the biomaterial and the repair process, we performed mCT and histological analysis on the tissue/biomaterial interface and the peripheral area of the implant. By using the former analysis, new bone formation was evident at different distances from the implant, which was more dramatic for the osteostatin-loaded biomaterials at both time periods studied (Figs. 1 and 2, upper panels). The osteoinductive effect of osteostatin was confirmed by quantification of % BV/TV and Pb.Tf. in these bone samples (Table 1). After 4 weeks of implantation, the unloaded materials were intact within the cavitary defect, surrounded by a fibrous cap ewhich
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Fig. 1. Tridimensional reconstruction images and saggital images by mCT (upper panels) and light microscopy (from hematoxylin/eosin stained sections) (lower panels) of the area surrounding the different implants (m), containing newly formed bone at different distances from each implanted material at 4 weeks after production of a cavitary defect in the rabbit femur.
was thicker in the case of SBA15-C8e in close proximity to the material, and abundant connective tissue formed the cortical surface of the callus (Fig. 1). Four weeks later, the materials were still separated from the bone marrow by the fibrous cap, but newly formed small trabeculae were evident in the area of the callus close
to the implant, which were more abundant in the vicinity of SBA15 (Fig. 2). Implantation of both osteostatin-loaded SBA15 and SBA15-C8 materials promoted tissue healing after 4 weeks, as shown by the appearance of abundant osteoid and new trabeculae all around the
Fig. 2. Tridimensional reconstruction images by mCT (upper panels) and light microscopy (from hematoxylin/eosin stained sections) (lower panels) of the area surrounding the different implants, containing newly formed bone at different distances from each implanted material at 8 weeks after production of a cavitary defect in the rabbit femur.
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Table 1 Bone structural parameters at different distances from the different implants tested at 4 and 8 weeks following production of a cavitary defect in the rabbit femur. SBA15 þ PTHrP (107e111)
4 weeks
SBA15
Distance (mm)
BV/TV (%)
Tb.Pf (mm1)
BV/TV (%)
Tb.Pf (mm1)
BV/TV (%)
Tb.Pf (mm1)
BV/TV (%)
Tb.Pf (mm1)
218 436
0.97 10.43
40.23 16.43
10.43 25.71
8.53 5.02
2.32 8.68
35.30 18.95
20.50 18.15
4.95 9.53
SBA15 þ PTHrP (107e111)
SBA15-C8
SBA15-C8þPTHrP (107e111)
8 weeks
SBA15
Distance (mm)
BV/TV (%)
Tb.Pf (mme1)
BV/TV (%)
Tb.Pf (mme1)
BV/TV (%)
SBA15-C8 Tb.Pf (mme1)
BV/TV (%)
SBA15-C8þPTHrP (107e111) Tb.Pf (mme1)
218 436
19.91 33.60
16.88 9.59
33.23 46.45
9.38 7.12
1.17 1.14
38.23 34.05
6.40 34.84
10.03 2.90
BV/TV, bone volume/total volume; Tb.Pf., bone trabecular pattern factor. Data correspond to a representative rabbit in each experimental group.
biomaterial (Fig. 1, lower panel). After 8 weeks, newly formed bone was predominant in the repairing area, as trabecular and cortical structures, and a hypertrophic scar appears toward the exterior of the implant; meanwhile, trabeculae were observed around the biomaterial in the vicinity of bone marrow (Fig. 2, lower panel). Moreover, at this time period, the fibrous cap was split, allowing osteoid tissue to be in contact with the biomaterial, in the osteostatin-loaded SBA15-C8 group (Fig. 2, lower panel). 3.2. Inmunohistochemistry RAM11, a specific antibody against rabbit monocytes/macrophages, was used to confirm the biocompatibility (no inflammation induction) of each tested material (Fig. 3). A low number of RAM11positive cells were detected in the vicinity of the implanted materials during the time of study. Macrophages (as RAM11-positive cells) appeared at the each material/tissue interface at 4 weeks post-implant. At 8 weeks, however, this response decreased in the SBA15-C8 group, related to fibrous cap formation (Fig. 3A and B). RAM11-positive macrophages in a row are clearly displayed at the
SBA15-C8/tissue interface at 4 weeks (Fig. 3C). These cells were still detected in the outer layer of the fibrous cap surrounding the implanted SBA15 material (Fig. 3D). Of interest, the presence of PTHrP (107e111) was found to dramatically decrease this macrophage infiltration associated to both types of tested biomaterials at the latter time period (Fig. 3A and B). A high proliferative activity (as PCNA positivity) was found in the wounded soft tissue, both in connective tissue and the vascular bed. After 4 weeks, the scar process maintains a high proliferative activity in all groups studied; so that the mean score values for PCNA positivity were: 10.7% and 6.6% for the same area around SBA15 and SBA15-C8 materials, respectively. Meanwhile, these values were: 17.6% and 20.9% for the corresponding areas close to each PTHrP (107e111)-loaded biomaterials, respectively. At 8 weeks, this proliferation rate dramatically decreased in all the specimens studied, so that PCNA staining remained detectable (<10% of cells/field) only in the case of peptide-loaded SBA15-C8 (Fig. 4). Nuclear immunostaining for the transcription factor Runx2 was detected in cells around both tested materials, SBA15 and SBA15-
Fig. 3. Changes in RAM11 immunostaining in the area surrounding the implanted materials during bone regeneration. Comparative score values at 4 weeks (A) and 8 weeks (B). *p < 0.001, SBA15-C8 þ PTHrP (107e111) vs. SBA15 þ PTHrP (107e111); ap<0.001, SBA15-C8 vs. SBA15-C8 þ PTHrP (107e111); bp < 0.05, SBA15 vs. SBA15 þ PTHrP (107e111); and c p < 0.05 SBA15 vs. SBA15-C8. Images show the presence of RAM11-positive macrophages (denoted by arrows) in the fibrous cap (FC) close to the implanted material (m) in representative samples from the SBA15-C8 group at 4 weeks (C) or from the SBA15 group at 8 weeks (D).
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Fig. 4. Representative PCNA immunostaining showing labeled cells (arrows) at the interface between material (m) (SBA15-C8 þ PTHrP (107e111)) and fibrous cap (FC) at 4 weeks (A) or in the split fibrous cap at 8 weeks (B) within the femoral defect in rabbit.
C8. It was similar in all the groups studied after 4 weeks during the repair process (Fig. 5A); whereas PTHrP (107e111) loading onto these materials, mainly in the case of SBA15-C8, significantly increased the number of Runx2-positive cells without affecting the cell staining pattern after 8 weeks (Fig. 5B). Abundant nuclear staining for Runx2 was evident in newly formed bone areas around PTHrP (107e111)-loaded SBA15-C8 biomaterial, compared to that observed close to peptide-unloaded SBA15-C8 material, at 8 weeks (Fig. 5C and D).
After 4 weeks, the number of osteopontin-positive cells was higher for PTHrP (107e111)-loaded SBA15-C8 biomaterial than for the other groups tested during the period of study (Fig. 6A). This positivity increased near SBA15, independent of the presence of the PTHrP peptide, at the end of study (8 weeks) (Fig. 6A and B). Similar changes were also observed in immunostaining score values for osteocalcin ea late osteoblast differentiation marker that is related to the mineralization processe in the bone healing tissue (Fig. 7AeC). Thus, at 4 and 8 weeks after implantation, these
Fig. 5. Runx2 immunostaining in the implant bone interface within the femoral defect in rabbits. Comparative score values at 4 weeks (A) and 8 weeks (B) bp < 0.001, SBA15 vs. SBA15 þ PTHrP (107e111); ap < 0.001, SBA15-C8 vs. SBA15-C8 þ PTHrP (107e111). Images show abundant positivity (arrows) for this transcription factor in cells around newly formed bone areas corresponding to SBA15-C8 þ PTHrP (107e111) (C), which contrasts to the poor staining in these areas close to this peptide-unloaded material (D), at 8 weeks. Blood vessels were evident in these microimages.
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Fig. 6. Changes in osteopontin immunostaining in the implant bone interface within the femoral defect in rabbits. Comparative score values at 4 weeks (A) *p < 0,05 SBA15 þ PTHrP (107e111) vs. SBA15-C8 þ PTHrP (107e111) and ap < 0,01 SBA15-C8 vs. SBA15-C8 þ PTHrP (107e111). At 8 weeks (B) ap < 0.001, C8-SBA15 vs. C8-SBA15 þ PTHrP (107e111). A representative image showing the pattern of osteopontin staining (denoted by arrows) corresponding to SBA15 þ PTHrP (107e111) at 8 weeks is shown (C).
staining values were significantly higher in this tissue around PTHrP (107e111)-loaded SBA15-C8 biomaterial than in the other groups studied. Interestingly, at 8 weeks, staining for this marker increased in both SBA15 groups tested and also in the PTHrP (107e111)-loaded SBA15-C8 group but not in the group containing the latter material alone (Fig. 7C). Considering the well characterized relationship between angiogenesis and osteogenesis [31], and the previously reported effect of osteostatin and/or the native C-terminal PTHrP fragment 107e139 on induction of the VEGF system in bone [16,20,21], we sought to assess VEGF immunostaining in the bone regenerating tissue close to the implanted materials. We found VEGF-positive cells in the healing tissue, including blood vessels and mesenchymal cells associated with high osteoid formation, only in the case of the PTHrP (107e111)-loaded biomaterials at 8 weeks (Fig. 8A and B). Sclerostin, the product of SOST gene that is specifically expressed in osteocytes, acts as an endogenous modulator of the canonical Wnt pathway and thus of bone formation [32,33]. Positivity for this marker in the repairing area showed a significant relationship to the presence of PTHrP (107e111) during the period of study (Fig. 9A and B). After 8 weeks, we observed a significant decrease for this positivity in osteocytes within this area close to the peptide-loaded SBA15-C8 biomaterial to values similar to those for the corresponding non-functionalized biomaterial (Fig. 9C). This might indicate that stabilization of the osteogenic process has occurred at this time with each type of tested biomaterial. To further support the hypothesis that this might be the case, we evaluated the presence of osteoclasts using
a specific TRAP anti-antibody. We found very few TRAP-positive polynucleated cells (osteoclasts) in all of the rabbit tissue samples studied; this was specially the case in the vicinity of SBA15-C8, mainly when PTHrP (107e111) was present (Fig. 10). Osteoclasts are cells derived from the monocyte/macrophage lineage. In fact, RAM11-positive cells were more abundant than TRAP-positive osteoclasts at 4 weeks, and similar to what was observed for the latter cells, the former cells dramatically decreased by the presence of the PTHrP peptide onto the implant (Figs. 3 and 10). 4. Discussion In our recent in vitro report [17], we showed that osteostatinloaded SBA15-based biomaterials display osteogenic features in osteoblastic cells. In this previous study, we found that osteostatin was released as a burst from both SBA15 and SBA15-C8 materials within the first 24 h in cell culture medium. However, even osteostatin remaining in these biomaterials and released at concentrations in the < nM range during prolonged incubations (up to 12 days) was efficient to affect osteoblastic function in vitro [17]. In this study, we also showed the higher efficacy of osteostatinloaded SBA15-C8 than that of its SBA15 counterpart to modulate the expression of some osteoblastic genes, namely osteocalcin (OC) and osteoprotegerin (OPG) [17]; likely as a consequence of the greater peptide adsorption to the external surface of the former functionalized biomaterial than in the case of SBA15, making it more accessible to the osteoblastic cell microenvironment. These in
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Fig. 7. Changes in osteocalcin staining in the area surrounding the different implants during bone regeneration within a femoral defect. Comparative score values at both time periods studied, 4 (A) and 8 weeks (B). *p < 0.02, C8eSBA15 þ PTHrP (107e111) vs. SBA15 þ PTHrP (107e111); ap < 0.001, C8-SBA15 vs. SBA15-C8 þ PTHrP (107e111); bp < 0.05, SBA15 vs. SBA15 þ PTHrP (107e111). #p < 0.01, between 4 and 8 weeks (C) for SBA15, SBA15 þ PTHrP (107e111) and SBA15-C8 þ PTHrP (107e111) implants. Image shows the presence of osteocalcinepositive cells in a representative tissue sample from the SBA15-C8 þ PTHrP (107e111) group at 8 weeks (D). Arrows denote the presence of osteocalcinpositive cells (osteoblasts).
vitro findings demonstrate the osteogenic features conferred by osteostatin coating onto SBA15-based materials, which prompted us to test them as putative biomaterials to promote bone regeneration in vivo. In the present study, we assessed the in vivo effects of PTHrP (107e111) delivered by SBA15 materials into a cavitary bone defect located in the epiphysis of the rabbit femur. This is an environment made up of trabecular bone and a minor cortical bone component
at the edge of the defect, thus unexposed to mechanical strains, which has been previously proven suitable for testing biomaterials [34,35]. Using this animal model, we have been able to show that these peptide-loaded bioceramics exert a clear bone regeneration action (as assessed by histological and mCT analysis and immunohistochemistry). Furthermore, our findings herein demonstrate that these PTHrP (107e111)-loaded biomaterials are highly osteoconductive and
Fig. 8. Representative image showing VEGF positivity in blood vessels (*) in regenerating bone (RB) (A) and in stromal cells within the bone marrow (arrows) close to the SBA15C8 þ PTHrP (107e111) biomaterial (m) (B) at 8 weeks. FC ¼ fibrous cap.
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Fig. 9. Changes in sclerostin immunostaining in the area surrounding the different implants in a femoral defect in rabbits. Comparative score values at 4 (A) and 8 weeks (B). a p < 0.001, C8-SBA15 vs. SBA15-C8þPTHrP (107e111); bp < 0.05, SBA15 vs. SBA15 þ PTHrP (107e111); #p < 0.02, between 4 and 8 weeks (C) for SBA15-C8þPTHrP (107e111). Image shows the presence of sclerotin-positive cells (osteocytes; arrows) in regenerating bone in a representative sample from the SBA15 þ PTHrP (107e111) group at 8 weeks (D).
osteoinductive by promoting infiltration of proliferating osteogenic precursor cells and connective tissue formation. In fact, the appearance of a thick fibrous cup induced by SBA15-C8 was significantly reduced by PTHrP (107e111) coating, associated with increased bone regeneration. The latter was further assessed by immunostaining for various osteoblast markers in the tissue
surrounding the implant. During the initial phase of the repair process (4 weeks), Runx2-positive cells increased in the newly formed trabeculae infiltrating the defect independent of the type of implant tested. This positivity decreased with time after implantation of both types of ceramics but remained high in the presence of PTHrP (107e111), related to an increased osteopontin and OC
Fig. 10. TRAP immunostaining in the healing bone area close to each implant after a femoral defect in rabbit. Comparative score values at 4 weeks (A) and 8 weeks (B). *p < 0.05, C8-SBA15 þ PTHrP (107e111) vs. SBA15 þ PTHrP (107e111); ap < 0.05, SBA15-C8 vs. SBA15-C8þPTHrP (107e111); cp < 0.02, SBA15 vs. SBA15-C8. Representative image showing the presence of TRAP-positive cells (arrows) in the SBA15-C8 group at 4 weeks.
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staining; the latter was specially so in the case of SBA15-C8 material. This is totally consistent with the crucial role of Runx2 for modulation of the latter osteoblastic differentiation genes [36]. In fact, this transcription factor has recently been shown to be a target of low-intensity ultrasounds to improve osteoblastic cell differentiation in delayed unions in humans [37]. This osteogenic effect of PTHrP (107e111) which extended the maturation phase during regeneration was associated with an increased cell proliferation (based on PCNA immunostaining) and an augmented number of sclerostin-positive osteocytes in cortical bone close to the implant. In addition, the PTHrP (107e111)-loaded biomaterials induced revascularization of the defect, related to an increased immunostaining for VEGF in the healing bone tissue. This is particularly interesting since systemic administration of PTHrP (107e139) has recently been shown to stimulate angiogenesis and the VEGF system in the regenerating tibia of mice with glucocorticoidinduced osteopenia [21]. In fact, current data underscore the key role of the latter system on the process of neoangiogenesis and new bone formation in the setting of bone healing [31,38]. Some monocytes/macrophages (based on RAM11 positivity) infiltrated the regenerating tissue around the implants during the period of study. For the functionalized SBA15 material, this moderate inflammatory response was transient, since RAM11 positivity was absent at 8 weeks post-implantation. Moreover, PTHrP (107e111) loading dramatically inhibited this response as well as the appearance of TRAP-positive osteoclasts in the vicinity of the implant. The latter was expected in view of the previously reported inhibitory effect of PTHrP (107e111) on osteoclastogenesis in vitro [8,9], and that of PTHrP (107e139) in vivo in the regenerating tibia of glucocorticoid-treated mice [21]. The present findings further support the notion that this PTHrP domain has anti-resorptive features, and suggest that PTHrP (107e111) inhibits monocyte/macrophage differentiation towards the osteoclastic lineage during bone regeneration in this bone healing model. 5. Conclusions The present report demonstrates that PTHrP (107e111)-coated bioceramics are efficient in favoring peri-implant bone regeneration in an experimental cavitary defect, as compared with the bioceramic alone. Moreover, SBA15 functionalization with C8 followed by PTHrP (107e111) coating allowed us to obtain a suitable biomaterial for local delivery of the peptide to promote bone regeneration in this setting. These findings could make these biomaterials as potential formulations for clinical application. Acknowledgments This study was supported by grants from Instituto de Salud Carlos III(PI050117, PI080922, and RETICEFRD06/0013/1002 and RD06/0013/1006), Ministerio de Educación y Ciencia (SAF2005e05254), Fundación de Investigación Médica Mutua Madrileña, Comisión Interministerial de Ciencia y Tecnología (CICYT, Spain)(MAT2008e736) and Comunidad Autónoma de Madrid (S2009/MATe1472). DL was supported by Fundación Conchita Rábago. Appendix Figures with essential color discrimination. Figs. 1e10 in this article have parts that are difficult to interpret in black and white. The full color images can be found in the online version, at doi:10. 1016/j.biomaterials.2010.07.103.
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