Bone 69 (2014) 154–164

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Original Full Length Article

Human recombinant cementum attachment protein (hrPTPLa/CAP) promotes hydroxyapatite crystal formation in vitro and bone healing in vivo Gonzalo Montoya a, Jesús Arenas b, Enrique Romo a, Margarita Zeichner-David c, Marco Alvarez d, A. Sampath Narayanan e, Ulises Velázquez a, Gabriela Mercado a, Higinio Arzate a,⁎ a

Laboratorio de Biología Periodontal, Facultad de Odontología, Universidad Nacional Autónoma de México, México Instituto de Física, Universidad Nacional Autónoma de México, México c Ostrow School of Dentistry, University of Southern California, USA d Laboratorio de Bioingeniería de Tejidos, Facultad de Odontología, Universidad Nacional Autónoma de México, México e School of Medicine, Department of Pathology, University of Washington, Seattle, USA b

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Article history: Received 10 June 2014 Revised 14 September 2014 Accepted 16 September 2014 Available online 28 September 2014 Edited by: David Burr Keywords: Cementum attachment protein Bone regeneration Hydroxyapatite Nanospheres Biomineralization

a b s t r a c t Cementum extracellular matrix is similar to other mineralized tissues; however, this unique tissue contains molecules only present in cementum. A cDNA of these molecules, cementum attachment protein (hrPTPLa/ CAP) was cloned and expressed in a prokaryotic system. This molecule is an alternative splicing of protein tyrosine phosphatase-like A (PTPLa). In this study, we wanted to determine the structural and functional characteristics of this protein. Our results indicate that hrPTPLa/CAP contains a 43.2% α-helix, 8.9% β-sheet, 2% β-turn and 45.9% random coil secondary structure. Dynamic light scattering shows that this molecule has a size distribution of 4.8 nm and aggregates as an estimated mass of 137 kDa species. AFM characterization and FE-SEM studies indicate that this protein self-assembles into nanospheres with sizes ranging from 7.0 to 27 nm in diameter. Functional studies demonstrate that hrPTPLa/CAP promotes hydroxyapatite crystal nucleation: EDS analysis revealed that hrPTPLa/CAP-induced crystals had a 1.59 ± 0.06 Ca/P ratio. Further confirmation with MicroRaman spectrometry and TEM confirm the presence of hydroxyapatite. In vivo studies using critical-size defects in rat cranium showed that hrPTPLa/CAP promoted 73% ± 2.19% and 87% ± 1.97% new bone formation at 4 and 8 weeks respectively. Although originally identified in cementum, PTPLa/CAP is very effective at inducing bone repair and healing and therefore this novel molecule has a great potential to be used for mineralized tissue bioengineering and tissue regeneration. © 2014 Elsevier Inc. All rights reserved.

Introduction Cementum extracellular matrix is similar to other mineralized tissues such as bone and dentin; however, this unique tissue contains specific molecules only expressed by cementocytes, cementoblats and their progenitor cells present in the periodontal ligament. These unique molecules, cementum attachment protein (CAP) and cementum protein 1 (CEMP1) are believed to regulate the biological activities of periodontal ligament cells [1–6]. The presence of these cementum-specific markers, their structural characterization and their patterns of gene expression has brought a better understanding of the molecular mechanisms that control cementum formation [1,7]. CAP was the first protein to be isolated and it was determined that it plays a role in cell ⁎ Corresponding author at: Laboratorio de Biología Periodontal, Facultad de Odontología, Universidad Nacional Autónoma de México, Ciudad Universitaria, México D.F. 04510, México. E-mail address: [email protected] (H. Arzate).

http://dx.doi.org/10.1016/j.bone.2014.09.014 8756-3282/© 2014 Elsevier Inc. All rights reserved.

recruitment and differentiation during cementum formation [8–12]. It was shown that it is expressed by dental follicle cells and promotes their adhesion and differentiation [12–15]. Furthermore, periodontal cells grown in culture in the presence of CAP are able to form a cementum-like mineralized tissue in vitro [16,17]. Recently we reported the identification of an alternatively spliced mRNA obtained from cell lysates of periodontal ligament and cementum-derived cells using a CAP specific antibody [4]. This mRNA encodes a polypeptide of 140 amino acids (GenBank Accession Number: AAR22554.1; GI:38503520). BLAST Analysis of the non-redundant NCBI protein database revealed that the N-terminal 125 amino acids of this mRNA was identical to a truncated isoform of 3-hydroxyacyl-CoA dehydratase 1/protein tyrosine phosphatase-like (proline instead of catalytic arginine) 1/PTPLa (GenBank Accession Number: AY455942.1; GI:38503519). This novel molecule was named PTPLa/CAP, and it is expressed in cementoblasts and some populations of periodontal ligament cells (probably cementoblast precursors) but not detected in other tissues like gingiva or bone [4].

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Cementoblasts are the principal cells responsible for the formation of cementum. In situ hybridization analysis demonstrated that expression of PTPLa/CAP coincides with cementum mineralization and it was expressed constitutively throughout the process of mineralization [4]. Furthermore, native CAP as well as human recombinant PTPLa/CAP (hrPTPLa/CAP) binds strongly to hydroxyapatite [4,18]. Biomineralization in general is a highly regulated process in vivo; it is believed that the hydroxyapatite present in mineralized tissues is assembled under the influence of a variety of extracellular matrix proteins, which act as nucleators, inhibitors, crystal growth regulators and/or scaffolds for mineral deposition [19]. The process of mineralization in cementum is regulated and controlled spatially and temporarily by the extracellular macromolecules synthesized by cementoblasts to form biocomposite structures. These observations suggest that the PTPLa/CAP may play a regulatory role during cementum formation and the biomineralization process. In the present study we wanted to further characterize hrPTPLa/CAP and to test the hypothesis that this protein promotes hydroxyapatite crystal nucleation in vitro and in vivo and therefore it has the potential to induce new bone formation which can heal surgically created critical-size defects (CSD) in rat calvaria. Materials and methods Expression and purification of recombinant human CAP Expression and purification of hrPTPLa/CAP was previously described [4]. Briefly, an E. coli BL21 (DE3) cell line containing a pDEST42/PTPLa/CAP construct was grown at 37 °C in LB medium containing 100 μg/ml ampicillin and 34-μg/ml chloramphenicol to an O.D. of 0.4 at 600 nm. Isopropyl-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM and the cell culture was incubated at 37 °C. Cells were harvested after 12 h by centrifugation and the cell pellet was lysed and processed as described elsewhere [2]. Purification was performed by Ni2+ affinity chromatography HisPrep FF 16/10 (GE Healthcare Bio-Sciences, Sweden), followed by HA-Ultrogel affinity chromatography (Sigma, St. Louis, MO, USA). Determination of protein purity and identity was performed by 12% SDS-PAGE combined with Coomassie blue staining and western blotting as described in detail elsewhere [2].

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Instruments, Ltd., UK) [22]. During experiments the temperature was held at 30 (0.1 °C) via a Peltier unit. Data analysis was performed using the Zetasizer Nano Family software package (V.7.10), (Malvern Instruments, Ltd., UK). Self-assembly of hrPTPLa/CAP studied by AFM. To investigate the selfassembly behavior of the hrPTPLa/CAP, solution samples were prepared by dissolving hrPTPLa/CAP in a 25 mM Tris–HCl buffer (pH 7.4) at different concentrations (25 μg, 50 μg and 100 μg). A drop (≈30 μl) of the solution samples was placed onto a silicon (1 1 1) monocrystal substrate and set for 5 min. The samples were dried by carefully holding a filter paper on the edges of the monocrystal substrate and then canned air-dried. The AFM images were obtained by operating a tapping-mode. Fields emission scanning electron microscopy (FE-SEM). Protein samples were preliminarily diluted as described above. Samples at a concentration of 25, 50 and 100 μg/μl were deposited onto silicon (111) monocrystal substrate under a biohazard safety cabinet to avoid any contamination after pipetting until the samples were canned air-dried. After the dehydration process samples were transferred to the vacuum process mounted onto metal stubs and treated by metal coating (i.e., gold/palladium) using vacuum controlled sputtering equipment in order to improve the electronic conductivity of the surface. The setting of sputtering process parameters, voltage and current, allows for the deposition of a thin layer which helps

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Characterization of hrPTPLa/CAP Characterization of hrPTPLa/CAP secondary structure, molecular sizing and self-assembly properties were done using the following methods: Circular dichroism spectroscopy. hrPTPLa/CAP protein was dissolved in 10 mM sodium phosphate buffer, pH 7.4 at 200 μg/ml. The protein concentration was calculated from the absorption at 280 nm using an extinction coefficient of 21,095 M−1 cm−1 and deduced from the amino acid sequence [20]. CD spectrum were recorded in thermostatted (25 °C) using quartz cells of 1-mm optical path length within a wavelength range of 190–260 nm using a Jasco J-710 spectropolarimeter. The molar ellipticity (θ) expressed in degrees cm2 dmol−1 was calculated on the basis of a mean residue of 110 g/mol. Five spectrum were accumulated to improve the signal to noise ratio. A baseline 10 mM sodium phosphate, pH 7.4 buffer solution was recorded separately and subtracted from each spectrum. The program Dicroprot was used to calculate secondary structure content which was performed with the Henessey & Johnson algorithm [21]. Dynamic light scattering. DLS measurements of the protein solution were obtained using a Zetasizer Nano S (Malvern Instruments, Ltd., UK) molecular sizing instrument which employs a 4 mw, 633 nm semiconductor laser as light source and NIBS technology (Malvern

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Fig. 1. (A) Circular dichroism indicates that hrPTPLa/CAP is mainly structured as α-helix (43.2%), β-sheet (8.9%), β-turn (2%) and random coil (45.9%) secondary structure. (B) Dynamic light scattering studies of hrPTPLa/CAP aggregates mainly at 4.8 nm and represents a molecule of an estimated mass of 137 kDa, as a result of cluster formation by nanospheres.

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to accurately scan the sample without altering the surface properties. SEM investigation is adapted to the specific properties of samples. In particular, samples under low vacuum conditions and spot were set to avoid any damage due to high local concentration of the beam energy onto the sample performed by the FE-SEM (JSM‐7600F). The average diameter of hrPTPLa/CAP protein morphology was measured from selected SEM micrographs by using open source image analysis software (Image J v.3.7; National Institutes of Health, U.S.A.). Mineralization experiments These experiments were conducted to elucidate the influence of hrPTPLa/CAP on the nucleation, morphology, micro and nanostructure of the calcium phosphate minerals. In vitro nucleation assays Calcium phosphates were crystallized by the slow and controlled chemical reaction between the calcium and phosphate ions (without organic matrix) in a semisolid medium at physiological pH and at room temperature. The semisolid crystallizing medium was prepared by using 2 ml of sodium metasilicate solution of specific gravity 1.06 g/ml, 1.08 ml of 1 M (85.7% purity) H3PO4, 2 ml of HEPES buffer 10 mM, pH 7.2. Protein solutions (10 μg/ml), were prepared in 10 mM HEPES pH 7.4 and mixed with the sodium metasilicate solution (SMS). The SMS solution was then allowed to polymerize incorporating the protein in the crystallizing medium. After gelation, the second reactant (100 mM CaCl2 in 10 mM HEPES buffer, pH 7.4) was added over the

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crystallizing medium. Gels were maintained at 37 °C for 7 days. As a negative control we have used bovine serum albumin [23]. Characterization of the mineral deposits To characterize the mineral deposits we used the following methods: Scanning electron microscopy. The morphology, microstructure and the elemental chemical composition of the calcium phosphates were examined by using a low vacuum scanning electron microscope (JSM 5600LV). The samples were fixed onto an aluminum specimen holder with carbon tape. The SEM elemental analysis was performed at 20–25 kV acceleration voltage and at 20–25 Pa of pressure in the specimen chamber [24,25] on different areas with different probe sizes. The images were obtained with backscattered electron signal (BSE) to know if the chemical composition of the crystal is homogeneous or non-homogeneous. The calcium to phosphate (Ca/P) ratio was calculated from the intensity of the peaks present in the EDS pattern and semi-quantitative analysis obtained by EDS (Noran X-ray microanalysis detector, model Voyager 4.2.3.) MicroRaman spectroscopic analysis. Crystals (experimental and controls) and spectrum were recorded with a MicroRaman DXR Thermo Scientific in the range from 1400 to 200 cm−1 using 532 nm wavelength excitation laser with a power of 10 mV. A holographic notch filter was used to filter out the Rayleigh radiation. The Raman radiation was dispersed using a 1800 groove/mm grating and detected by a Peltier cooled charge-coupled device array.

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Fig. 2. Human recombinant PTPLa/CAP protein assembly as observed by tapping mode AFM. (A) Full length hrPTPLa/CAP protein aggregates into chains of connected nanospheres organized into string-like structures (B) Three-dimensional view of the nanospheres revealed a supramolecular organization showing collinear arrays of hrPTPLa/CAP nanospheres resembling a globular organization with no spaces in between the nanospheres. (C) Human recombinant PTPLa/CAP forms a rod-like structures conformed by nanospheres. (D) Three-dimensional view shows the organization of nanospheres and rod-like structure around an empty space that resembles a honeycomb-like organization of hrPTPLa/CAP protein.

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Transmission electron microscopy. A JEM2010 FEG with 0.19 nm resolution was used to analyze the calcium phosphates. For TEM studies, the calcium phosphate precipitates were suspended in isopropyl alcohol and scattered by ultrasonic bath, then a drop was placed on carbon Formvar-coated copper grids of 300 mesh. High resolution transmission electron microscopy (HRTEM). Morphology and microstructure were observed by using a High Resolution Transmission Electron Microscope (HRTEM) JEOL JEM-2100F with an accelerating voltage of 200 kV and resolution of 0.19 nm. Digital Micrograph Software was used in order to analyze the crystal structure of the samples. The interplanar distances measured were compared to standard hydroxyapatite (JCPDS 44-0778. 2001), [26]. Atomic force microscopy. Atomic force microscopy (AFM) was used to determine the topography and homogeneity of the crystals formed as a result of hrPTPLa/CAP (10 μg/ml) induction in metasilicate gels. The examination was performed with an AFM (JSPM 4210) with a silicon tip (NSC 15), cantilever length 125 μm, with a constant applied force (40 N/m) and a resonance frequency of 325 kHz rate in dry samples [27]. Measurements were performed at room temperature. In vivo mineralization studies To determine the potential of hrPTPLa/CAP to induce mineral formation In Vivo we used a rat calvaria critical-size defect (CSD) healing model. Surgical procedures Male Wistar rats aged 7–8 weeks and weighting 250–300 g were obtained from the Facultad de Medicina Vivarium (Universidad

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Nacional Autónoma de México), and acclimatized at the animal research facility for 7–14 days before the start of the experiments. All animal procedures were approved by the, Ethical and Animal Care and Use Committee (Facultad de Odontología, Universidad Nacional Autónoma de México). Eight rats were used for each group; 1). Control group — nothing added; 2). Protein control group — gelatin matrix scaffold and 3). Experimental group — gelatin matrix scaffold with hrPTPLa/ CAP. Animals were anesthetized as described elsewhere [28], the surgical site on the dorsal surface of the cranium was shaved and cleaned with disinfectant. A 3-cm midline incision was made over the calvaria and the periosteum was completely cleared from the surface of the cranial bone by scraping. A 9-mm craniotomy defect was created with a trephine attached to an electrical drill under copious saline irrigation. The calvaria disk was then removed with a blunt surgical probe. For implantation, gelatin matrix sponges were cut to the size of 9 mm diameter, and 10 μg of hrPTPLa/CAP was placed on top of the gelatin matrix and left to settle for 1 h and dried in a lyophilizer. Empty gelatin matrix was used as a control. To confirm that the 9 mm defect was critical-sized, we also created a defect without implanting a scaffold, and examined this condition at 4 and 8 weeks also. The surgical site in all conditions was covered by a thin gelatin membrane to avoid contact of the periosteum with the surgical site. The skin was closed using 4–0 silk sutures [28]. Animals were euthanized with carbon monoxide after 4 and 8 weeks. Histological procedures and bone regeneration analysis Calvarias were fixed overnight in 10% formaldehyde as described elsewhere [28]. The specimens were decalcified with 10% EDTA, pH 7.4, dissolved in 0.5% formaldehyde at 4 °C for 5 weeks, dehydrated in a graded alcohol series, embedded in paraffin, and sectioned at 5 μm

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Fig. 3. FE-SEM shows hierarchical self-assembly organization of hrPTPLa/CAP. (A) Individual PTPLa/CAP nanospheres along with these nanospheres that organize themselves into nanostrings or fiber-like structures by alignment and fusion of nanospheres. (B) The nanospheres show an average size of 27 nm. (C) FE-SEM shows well organized nanostrings-like structures forming a net-like structure. (D) Higher magnification indicates the fusion of nanospheres in a larger molecular organization to form a fiber-like structure by hrPTPLa/CAP. These structures show an average size of 18 nm (D).

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G. Montoya et al. / Bone 69 (2014) 154–164 Ca/P Ratio: 1.6

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Ca/P Ratio: 1.0

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Fig. 4. SEM images of spheres-like structures formed in vitro by hrPTPLa/CAP (inset indicates CA/P ratio of 1.6) (A). (B) SEM image of an isolated sphere-like structure showing the formation of crystals in the form of tagliatelle-like morphology (see inset in B). (C) An open microsphere shows different phases; a core representing immature mineral phase (*), an interphase between immature and organized crystals irradiating from this well-defined phase (arrow). Irradiating crystals are observed emerging from this phase (see inset in C). (D) Control platelike crystals formed in the controls, using BSA as a blank protein. Ca/P ratio is of 1.0 (see inset in D).

thick. The calvarias were sectioned perpendicular to the sagittal suture. Three central sections per defect stained with hematoxylin and eosin were used for the histological/ histomorphometric analysis. Additionally, three central sections were stained with Masson's trichrome for cross-reference. The mineralized tissue area was normalized to the total tissue area and computed by using Image Pro Plus Software. Analysis of the defects with tissue was performed for all conditions via light microscopic analysis.

Molecular analysis of the filled defect Expression of bone-related molecules was also analyzed in the new formed bone-like tissue in rat calvaria by double-immunofluorescence staining using mouse monoclonal antibody against human osteopontin (OPN), human bone sialoprotein (BSP) and human STRO-1, rabbit polyclonal antibody against human OCN (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and against human PTPLa/CAP (produced in house). Tissues were fixed, decalcified, embedded in paraffin, sectioned and mounted on glass silanized slides as described elsewhere [2]. Sections were pretreated with 10% BSA in PBS for 1 hr at room temperature and incubated with 1:100 diluted primary antibodies for 12 h at 4 °C. Sections were then incubated with 1:100 diluted Alexa-Fluor-488-conjugated anti-mouse IgG (Molecular Probes; Eugene, OR) and Alexa-Fluor-594-conjugated anti-rabbit IgG (Molecular Probes) as secondary antibodies for 1 h at room temperature. Samples were evaluated using a fluorescent microscope (Axioskope 2, Carl Zeiss, Germany) with the appropriate filter combinations. Negative controls were achieved by omitting the primary antibody or by incubating with normal rabbit or mouse serum.

Statistical analysis Eight animals were used for control and experimental groups and values are expressed as mean ± S. E. Multiple comparisons between treatment groups were made with the Neuman-Keuls post-hoc test with a two-way analysis of variance (ANOVA). P b 0.05 was considered statistically significant. Statistical analyses were performed with Sigma Stat V 3.1 software (Jandel Scientific Ashburn, VA).

Fig. 5. As can be seen, the Raman spectrum of crystals formed by hrPTPLa/CAP has welldefined peak at 961 cm−1 that may be ascribed to the presence of phosphate, one of the constituents used to form mature hydroxyapatite. The more crystalline the structure, the narrower the band is.

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Results Characterization of hrPTPLa/CAP Circular dichroism of hr/PTPLa/CAP showed the presence of spectrum with two negative maximum values near 210 and 218 nm. This spectrum is similar to other proteins mainly composed by alpha-helix. CD spectrum analysis revealed 43.2% α-helix, 8.9% β-sheet, 2% β-turn and 45.9% random coil (Fig. 1A). This determines that the secondary structure present is mainly composed of random coil and α-helix structure. The theoretical isoelectric point of hrPTPLa/CAP was determined to be 6.37. Dynamic light scattering analysis revealed that hrPTPLa/CAP forms aggregates with radium of 4.8 nm. Such aggregates are contributed by an estimated mass of 137 kDa (Fig. 1B). Analysis of these aggregates was further done using AFM and FE-SEM, which showed that hrPTPLa/CAP protein forms spheres-like structures at the nanometer scale. These nanospheres are not isolated but rather connected to each other forming organized aggregates (Figs. 2A–B). AFM scans at high resolution, revealed that the original nanospheres are 27 nm average size and they gradually fused together to form a continuous string-like feature. These strings align together forming an ultrastructure similar to a “net-like feature” (Figs. 2C–D). The shape of the nanospheres was not always rounded; moreover the spheres appear deformed and often flattened at the areas where they connect with each other (Fig. 2B). The results obtained from FE-SEM analysis shows that hrPTPLa/CAP nanospheres have an average size of 27 nm which confirms the AFM results (Figs. 3A–B). They also indicate a hierarchical self-assembly organization of hrPTPLa/CAP from nanospheres into nanostrings by

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alignment and fusion of the original nanospheres. In general, the appearance of the nanospheres is homogeneous although some large components representing nanostrings are also present (Figs. 3C–D). Taken all together the data indicates that hrPTPLa/CAP is a protein with a high percentage of random coil structure (structural disorder), that it is an acidic protein and that it self-assembles into nanospheres which then form aggregates resembling nanostrings. It has been shown that intrinsically disordered proteins (IDPs) are multifunctional and can have hydroxyapatite binding properties, as is the case of SIBLING and HMGI(Y) [29,30]. It has also been shown that acidic proteins perform different roles in hydroxyapatite prismatic formation; therefore it can be expected that hrPTPLa/CAP might have some function in hydroxyapatite crystal formation. Furthermore, the self-assembly of this protein suggests the formation of functional building blocks that can facilitate oriented hydroxyapatite crystal formation by providing the ultrastructural framework. Can hrPTPLa/CAP induce formation of hydroxyapatite? Human recombinant PTPLa/CAP was utilized for nucleation studies as described in the Methods section. Results from these experiments indicate that this protein promotes hydroxyapatite formation and growth after 7 days as compared to protein-free controls and other proteins like BSA used as protein control. SEM analysis of the mineral formed showed the formation of spherical particles containing needle-like substructures with a diameter of 350 ± 6.0 μm (Fig. 4A). At higher magnification these structures represented aggregates of densely packed elongated needle-like crystals (Fig. 4B). These spherulites contained a core which

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Fig. 6. AFM shows the three-dimensional morphological disposition of the hydroxyapatite crystals formed by hrPTPLa/CAP. (A) Crystals revealed a longitudinal arrangement, perpendicular to the surface of the crystal axis. (B) The arrangement showed the features of valleys and promontories. (C) The crystal agglomeration favored the formation of crystalline plaques with a prism-like pattern in a wall-like formation. (D) Higher magnification shows the top of the irregular prism-like structures with a sphere-like end. Individual prism-like crystals showed an average width of Å 240 and were 116 nm long.

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225

115

[110]

0.119 nm 0.132 nm

Fig. 7. HRTEM images of the crystals shows the inter-planar distances near to planes and
(225), of the hydroxyapatite crystalline phase. The 115 Fast Fourier transform (FFT) of the crystal is indicated (Inset).

consisted of an immature mineral phase and the crystals (needle-shaped) emerged radially from this core with a length of 120 ± 0.09 μm (Fig. 4C). The Ca/P ratio of this immature mineral material was determined by EDS analysis. The Ca/P ratio of the core was of 1.2 ± 0.03. An interphase between the core and the emerged crystals was clearly observed. The crystals emerging adjacent to this interphase revealed a Ca/P ratio of 1.59 ± 0.06 which is similar to the ratio of 1.50 found in bone hydroxyapatite. The low Ca/P ratios of apatites under physiological conditions are widely believed to result from structural calcium deficiencies. An explanation of Ca deficiency in hydroxyapatite was based on the concept of an interlayering of octacalcium phosphate and stoichiometric hydroxyapatite [31–33]. The morphological features observed in these arrangements are in agreement with the apatite crystals found in human woven bone and mineralized cementum [19]. Negative control using BSA as blank protein produced an EDS spectrum of 1.0 ± 0.08 (Fig. 4D). Further confirmation of the crystals being hydroxyapatite was done using Raman

spectroscopy. A predominant band at 960 cm−1 in the Raman spectrum was consistent with the presence of hydroxyapatite (Fig. 5). Additional characterization of the crystals formed in the presence of hrPTPLa/CAP was done using AFM. The results obtained showed a sequence of the three-dimensional disposition of the hydroxyapatite crystals with a longitudinal arrangement perpendicular to the surface of the crystal axis (Figs. 6A–B). This arrangement showed the features of peaks and valleys with a size range of 70 nm. This crystal agglomeration favored the formation of crystalline plaques with a prism-like pattern. Individual prism-like crystals showed an average width of Å 240 and were 116 nm μm long (Figs. 6 C–D). Analysis using HRTEM shows the interplanar distance near to planes 115 and 225 in the direction of the hydroxyapatite crystalline phase (Fig. 7; JCPDS 44-0778) [26]. The Fast Fourier transform (FFT) of the crystal is indicated (Inset). Taken together all these results show that hrPTPLa/CAP can induce formation of hydroxyapatite crystals in a highly ordered structure consisting of spherulites containing a large number of hydroxyapatite long needles originating from an immature mineral center. Can hrPTPLa/CAP induce bone formation in vivo? To answer this question we used the model of rat calvaria criticalsized defects. Gelatin matrix scaffolds containing hrPTPLa/CAP or blank gelatin matrix scaffolds were implanted into rat calvaria criticalsized defects and samples were collected after 4 and 8 weeks postsurgery. After 4 weeks post-surgery, control defects (no matrix was placed) displayed growth of a thin connective fibrous tissue layer without evidence of bone formation (Fig. 8A). Defects implanted with gelatin matrix scaffolds were bridged with a thicker layer of fibrous connective tissues (Fig. 8B). Experimental scaffolds of gelatin matrix containing hrPTPLa/CAP showed a partial filling of the cranial defect with a mineralized type of tissue resembling the calvarial tissue present at the edges of the defect (Fig. 8C). Similar results were found after 8 weeks; the newly formed bone-like tissue shows morphological characteristics of normal bone with osteocytes embedded in their lacuna and osteoblasts lining the outer edge of the bone tissue (Figs. 9A–B). Cranial defects treated with gelatin matrix scaffolds containing hrPTPLa/CAP showed almost a complete filling of the cranial defect with bone-like tissue (Fig. 9C). No evidence of inflammatory response in the experimental or control conditions was observed. In order to quantitatively evaluate bone

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Fig. 8. Histological Sections stained with H&E after 4 weeks of treatment with hrPTPLa/CAP in a rat calvaria critical-size defect. (A) Control empty defect. Margins of the defect show connection with a dense fibrous connective tissue. (B) Control with gelatin matrix shows the defect occupied for dense fibrous connective tissue. (C) Gelatin matrix embedded with hrPTPLa/ CAP shows formation of bone tissue in the defect. All photomicrographs were taken at 100×. Arrowheads show the border of the calvarial defect.

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Fig. 9. Histological sections stained with H&E after 8 weeks of treatment with hrPTPLa/CAP in a rat calvaria critical-size defect. (A) Empty defect and (B) gelatin matrix-treated defect show connection of the defect's borders by a dense fibrous connective tissue. (C) Experimental defect treated with gelatin matrix embedded with hrPTPLa/CAP show bone formation and a space occupied by dense connective fibrous tissue and osteoid tissue. All photomicrographs were taken at 100×. Arrowheads show the border of the calvarial defect.

formation, histomorphometric analysis was performed using H&E stained sections. Samples treated with gelatin matrix scaffolds containing hrPTPLa/CAP demonstrated a 73% ± 2.19% and 87% ± 1.97% new bone formation at 4 and 8 weeks respectively. This was statistically significant as compared to the gelatin only matrix scaffolds and the cranial defect not treated (Fig. 10). Control specimens, blank defect and gelatin matrix alone, were filled with fibrous tissue only. Molecular analysis of the filled defect In order to assess the molecular identity of the filled defect, the presence and expression of bone-related molecules was analyzed by double-immunofluorescence. OPN was present in osteocytes and cells inside bone marrow spaces (Fig. 11A). BSP was present in cells surrounding mineralized structures and osteoblasts facing the mineralized front of bony tissue as well as osteocytes immersed into the mineralized matrix (Figs. 11B–C). OCN was detected in a similar distribution as BSP (Figs. 11F–G) and both co-localized in the same cellular structures (Fig. 11H). PTPLa/CAP was expressed in osteocytes and cells into the bone marrow (Fig. 11K). STRO-1 showed similar spatial distribution

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Fig. 10. Histomorphometric analysis of calvarial defects treated with hrPTPLa/CAP. The graphic shows that after 4 and 8 weeks post-surgery, the critical-sized calvarial defects, showed a 73% ± 2.19% and 87% ± 1.97% new bone formation.

(Fig. 11L). However, only a few osteocytes co-expressed both PTPLa/ CAP and STRO-1 (Fig. 11M). Controls using pre-immune mouse and rabbit serum were negative. (Figs. 11D, I, N). Discussion In the present study, we provided further characterization of hrPTPLa/CAP secondary structure, self-assembly properties and biological capabilities of this protein. AFM and FE-SEM studies indicate that this protein self-assemblies into nanospheres with sizes ranging from 7.0 nm to 27 nm in diameter. These nanospheres aggregate with each other in a string-like form to acquire a supra-molecular mesh-like structure. Evidence supporting the fact that the nanospheres aggregate in larger particles could be appreciated in the light scattering data. The fact that there appears only one species with an estimated mass of 137 kDa could be a result of cluster formation by nanospheres, moreover, in the absence of electrostatic repulsion, inter-particle hydrophobic interactions may lead to particles that can rest on top of each other thereby creating multilayers of particle aggregates [34,35]. We propose that these elongated structures provide guidance for the highly anisotropic growth of apatite crystals in bone/cementum. We suggest that hrPTPLa/CAP aggregates adopt a functional role which facilitates the nucleation, growth and direction of hydroxyapatite crystals. We can argue that the microspherical structures obtained in the nucleation experiments, containing an immature mineral phase in the center of the sphere, indicates that it acts as a “nucleus” and then the hydroxyapatite crystals irradiate from this solid-state nucleation center. The exact mechanism through which hrPTPLa/CAP regulate this process is not completely clear, however, it is known that aminoacid segments of PTPLa/CAP are of hydrophobic nature, which can interact directly with hydroxyapatite [36]. Also the nanospheres supramolecular structure may promote calcium phosphate deposition by concentrating charge at the hydrophobic surface and therefore acting as a nucleation template. The secretion of hydrophobic proteins into the extracellular space could provide space for mineral deposition. Circular Dichroism analysis revealed that hr/PTPLa/CAP is predominantly composed of random coil and alpha-helix structures, therefore this protein can be considered an intrinsically disordered protein [29]. These classes of proteins are multifunctional and allow proteins to have diverse binding properties including those associated with biomineral formation [37–40]. Bioinformatics studies have shown that

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α-OPN

b

α -BSP

BM

b

BM

b

OCT

α-BSP

OCT

OB

b

OCT

b

MERGE OB

OCT

b

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I b

-C J H&E

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OCT

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BM

M

BM

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-C E H&E

b

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α-STRO-1

BM

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OCT

G

α-PTPLa/CAP

OCT

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OCT

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b

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OCT

A α-OCN

MERGE

N

-C O

Fig. 11. Double staining and co-localization of bone-related proteins (OPN, BSP, OCN), STRO-1 and hrPTPLa/CAP in calvarial defects treated with hrPTPLa/CAP. Osteopontin (OPN) localizes the osteocytes and bone marrow (A). Bone sialoprotein (BSP) and osteocalcin (OCN) is expressed by osteoblasts (F and G). PTPLa/CAP localizes to osteocytes and bone marrow spaces (K) and STRO-1 localizes to some subpopulations of osteocytes and bone marrow space (L). Co-localization of OPN and BSP indicates homologous distribution in osteocytes and bone marrow spaces (C). Co-localization of PTPLa/CAP and STRO-1 co-localize in subpopulations of osteocytes and bone marrow spaces (M). Representative controls using normal rabbit and mouse serum are negative (D, I and N). Sections stained with H&E are for morphological orientation (E, J and O). All photomicrographs were taken at 40×. OCT: osteocytes; BM: bone marrow; OB: osteoblasts.

intrinsically disordered and aggregation-prone domains exist within the diverse set of human extracellular matrix protein sequences [40]. These domains are believed to be responsible for observed matrix assembly and hierarchal ordering of the extracellular matrix. In addition hrPTPLa/CAP showed a theoretical isoelectric point of 6.37 and acidic proteins perform different roles in crystal formation. These features suggest a role for hrPTPLa/CAP during cementum/bone formation as regulators of mineral formation by nucleation of hydroxyapatite and/or control of crystal growth as has been shown for other self-associative proteins involved in the process of biomineralization [41–44]. Recombinant human PTPLa/CAP possesses functional biological activity in terms of self-assembly and hydroxyapatite nucleation. Our studies also strongly suggest that the hydroxyapatite crystal formation by hrPTPLa/CAP might be assembled through an immature mineral phase. Thus, hrPTPLa/CAP appears to be responsible for regulating the transformation of an immature mineral phase to crystalline hydroxyapatite. Overall matrix intervention during the nucleation and growth process gives rise to composite materials like bone and cementum with functionalized mechanical properties [45]. Human recombinant PTPLa/ CAP protein could assemble ordered nuclei from nano- to micro-scales which in turn serve as a basis for the hierarchical structure of biomineral formation [46]. From the observations in this study, the nanospheres assembly and their interrelationship with calcium phosphate, highlight the potential of this protein to control the nucleation, morphology and growth of hydroxyapatite crystals in orchestration with other molecules present into the extracellular matrix of cementum and bone. Our findings also demonstrate that hrPTPLa/CAP undergoes aggregation therefore suggesting the potential for hierarchical selfassembly into elongated structures that could provide guidance for the highly anisotropic growth of apatite crystals in bone and cementum. The fibrous-like apatite nanocrystals observed in our experiments are a product of protein-guided growth, suggesting that the protein itself

may provide a dynamic scaffold to form elongated structures directing the growth of mineral. The possibility of generating elongated fibrillar structures and net-like organic matrices through cooperative selfassembly of hrPTPLa/CAP in vitro, appears to be a valuable model to understand the remarkable anisotropic growth that apatite crystals undergo in vivo. The intense structural analysis of hrPTPLa/CAP performed by FESEM, shows that the protein organizes into structural fiber network which make a refined and higher levels of resolution respect to the supramolecular organization of the protein [47]. Using an established craniofacial defect model, hrPTPLa/CAP showed significantly greater bone fill compared as compared to controls, indicating the potential of hrPTPLa/CAP in craniofacial bone regeneration/ augmentation. Calvarial critical size defects do not regenerate per se during the experimental lifetime of the animals and allow the evaluation of biomaterials that may induce bone regeneration as well as the adverse effects [48,49]. Human recombinant PTPLa/CAP showed 73% ± 2.19% and 87% ± 1.97% new bone formation at 4 and 8 weeks respectively when compared with the absorbable gelatin sponge and sham-surgery controls. Despite advances in our understanding of the multiple bio-functions of the cementum attachment protein from previous studies [16,50,51], to the best of our knowledge, no study has yet been conducted to report its effects on bone regeneration in the context of injured tissue. The present study clearly demonstrates that hrPTPLa/ CAP induces bone formation and therefore tissue regeneration and the nature of the mineralized tissue resembles bone at the histological and molecular levels. In the future our results can motivate further study of the cellular, molecular, temporal and spatial events that occur after the application of PTPLa/CAP. This may lead to the development of new strategies to enhance bone regeneration including craniofacial defects, treatment of fractures, bone augmentation for the placement of dental implants and for the regeneration of periodontal structures lost due periodontal disease.

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It is conceivable that hrPTPLa/CAP-induced bone regeneration is due to the induction of neovascularization and the subsequent recruitment of MSCs, since this protein has been shown to be expressed by paravascular stem cells in the periodontal ligament, and these cells are the suspected progenitors of osteoblasts and cementoblasts and express STRO-1 [52,53]. Our results support this statement since both PTPLa/ CAP and STRO-1 are co-expressed by cells located into the bone marrow spaces. Therefore this protein may promote cell differentiation and cell commitment toward different phenotypes and make this protein a strong candidate to regulate the mineralization process during bone regeneration. One of the putative mechanisms of the effect of PTPLa/CAP is through its ability to increase osteoblast differentiation and mineralization, thus contributing to osteoblastic function [54–56]. Nowadays, most bone regenerative strategies have focused on BMP-2, a potent osteoinductive molecule [57,58]. However, concerns about the use of supra-physiologic doses required for effective bone regeneration, as well as adverse effects such as heterotopic bone formation, idiopathic hematomas and high cost associated with such large doses [59–62], uphold the challenge to identify molecules effective in inducing bone healing. PTPLa/CAP, although originally identified in cementum, can be one of those proteins; this novel molecule has great potential for mineralized tissue bioengineering and tissue regeneration.

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