Nucleation and growth inhibition of biological minerals by cementum attachment protein-derived peptide (CAP-pi).

Biomineralization is a highly regulated process where proteins/peptides-crystal interactions contribute to the shaping, phasing and aggregation of minerals. We have identified and synthesized a cementum attachment protein-derived peptide (CAP-pi), which corresponds to amino acids 40-53 of the N-terminal CAP domain (MASSDEDGTNGGAS) and its phosphorylated variant (MASpSpDEDGTNGGASp) (CAP-pip). The peptide is composed of polar and negatively charged amino acids, which are disordered, according to in silico analysis. Our results show that CAP-pi inhibits hydroxyapatite (HA) formation and growth. However, it possesses low capacity to inhibit calcium oxalate crystal growth. CAP-pip showed a stronger inhibitory effect on the formation and growth of HA. As well as a high capacity to inhibit calcium oxalate monohydrate growth, mainly due to adsorption on specific growth faces. Small peptides have many advantages over the full-size protein, including low-cost production and modulation characteristics that allow for structural changes. Our findings suggest that CAP-pip-derived peptide could possess therapeutic potential to prevent or treat pathological calcifications such as renal stones and vascular calcification.

Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/ß-catenin Signaling in Human Oral Mucosa Stem Cells.

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/ß-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/ß-catenin pathway. CEMP1-p4 stimulation upregulated the expression of ß-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of ß-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the ß-catenin signaling cascade.

Cementum protein 1-derived peptide (CEMP 1-p1) modulates hydroxyapatite crystal formation in vitro.

A cementum protein 1-derived peptide (CEMP1-p1) consisting of 20 amino acids from the CEMP1's N-terminus region: MGTSSTDSQQAGHRRCSTSN, and its role on the mineralization process in a cell-free system, was characterized. CEMP1-p1's physicochemical properties, crystal formation, and hydroxyapatite (HA) nucleation assays were performed. Crystals induced by CEMP1-p1 were analyzed by scanning electron microscopy, Fourier-transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM), and atomic force microscopy. The results indicate that CEMP1-p1 lacks secondary structure, forms nanospheres that organize into three-dimensional structures, possesses affinity to HA, and induces its nucleation. CEMP1-p1 promotes the formation of spherical structures composed by densely packed prism-like crystals, which revealed a Ca/P ratio of 1.56, corresponding to HA. FTIR-ATR showed predominant spectrum peaks that correspond and are characteristic of HA and octacalcium phosphate (OCP). Analysis by XRD indicates that the crystals show planes with a preferential crystalline orientation for HA and for OCP. HRTEM showed interplanar distances that correspond to crystalline planes of HA and OCP. Crystals are composed by superimposed lamellae, which exhibit epitaxial growth, and each layer of the crystals is structured by nanocrystals. This study reveals that CEMP1-p1 regulates HA crystal formation, somehow mimicking the in vivo process of mineralized tissues bioformation.

Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.

The use of recombinant proteins has revolutionized the development of biologic pharmaceuticals; however, they are not free of complications. Some have very high molecular weight, some demonstrate in vivo instability, and the high cost of producing them remains a major problem. On the other hand, it has been shown that peptides derived from active domains keep their biologic activity and can trigger events, such as osteogenesis and bone regeneration. Small peptides are advantageous because of their ease of synthesis and handling and their low immunogenic activity. The purpose of this study was to investigate the functions of a synthetic peptide, cementum protein 1-peptide1 (CEMP-1-p1), both in vitro and in vivo. Our results show that CEMP-1-p1 significantly enhanced the proliferation and differentiation of human periodontal ligament cells toward a mineralizing-like phenotype, as evidenced by increasing alkaline phosphatase (ALP)-specific activity and osterix, runt-related transcription factor (RUNX)-2, integrin binding sialoprotein, bone morphogenetic protein-2, osteocalcin, and cementum protein (CEMP)-1 expression at mRNA and protein levels. In vivo assays performed through standardized critical-size calvarial defects in rats treated with CEMP-1-p1 resulted in newly formed bone after 30 and 60 d. These data demonstrate that CEMP-1-p1 is an effective bioactive peptide for bone tissue regeneration. The application of this bioactive peptide may lead to implementing new strategies for the regeneration of bone and other mineralized tissues.-Correa, R., Arenas, J., Montoya, G., Hoz, L., López, S., Salgado, F., Arroyo, R., Salmeron, N., Romo, E., Zeichner-David, M., Arzate, H. Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.

Human recombinant cementum attachment protein (hrPTPLa/CAP) promotes hydroxyapatite crystal formation in vitro and bone healing in vivo.

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% ß-sheet, 2% ß-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.

Bone regeneration in rat cranium critical-size defects induced by Cementum Protein 1 (CEMP1).

Gene therapy approaches to bone and periodontal tissue engineering are being widely explored. While localized delivery of osteogenic factors like BMPs is attractive for promotion of bone regeneration; method of delivery, dosage and side effects could limit this approach. A novel protein, Cementum Protein 1 (CEMP1), has recently been shown to promote regeneration of periodontal tissues. In order to address the possibility that CEMP1 can be used to regenerate other types of bone, experiments were designed to test the effect of hrCEMP1 in the repair/regeneration of a rat calvaria critical-size defect. Histological and microcomputed tomography (µCT) analyses of the calvaria defect sites treated with CEMP1 showed that after 16 weeks, hrCEMP1 is able to induce 97% regeneration of the defect. Furthermore, the density and characteristics of the new mineralized tissues were normal for bone. This study demonstrates that hrCEMP1 stimulates bone formation and regeneration and has therapeutic potential for the treatment of bone defects and regeneration of mineralized tissues.

Cementum protein 1 (CEMP1) induces differentiation by human periodontal ligament cells under three-dimensional culture conditions.

PDL (periodontal ligament) is a source of multi-potent stem cells in humans and their differentiation potential to a cementoblast and osteoblast phenotypes has been shown. Tissue construction from PDL-derived cells could be considered as a valuable technique for periodontal regenerative medicine. On these basis, we determined the role of CEMP1 (cementum protein 1) as a factor to induce differentiation of human PDL cells in a 3D (three-dimensional) fashion. Human PDL cells were grown in an RCCS (rotary cell culture system) D-410 RWV (rotating wall vessel) bioreactor, and maintained in either experimental (CEMP1 2.5 µg/ml) or control media during 4 weeks. Cell proliferation in the presence of CEMP1 was determined. The tissue-like structure formed was analysed histologically, stained with Alizarin Red and Alcian Blue. ALP (alkaline phosphatase)-specific activity, immunostaining, RT-PCR (reverse transcription-PCR) and Western blotting were performed to determine the expression of BSP (bone sialoprotein), enamel [AMBN (ameloblastin) and AMEL (amelogenin)], cementum [CAP (cementum attachment protein) and CEMP1] and cartilage-related proteins (Sox9, aggrecan, types II and X collagens). Our results show that hrCEMP1 (human recombinant CEMP1) promoted cell proliferation by human PDL cells in 3D cultures and induced the formation of a tissue-like structure resembling bone and/or cementum and material similar to cartilage. The addition of hrCEMP1 to the 3D human PDL cell cultures increased ALP-specific activity by 2.0-fold and induced the expression of markers for the osteogenic, cementogenic and chondrogenic phenotypes at the mRNA and protein levels. Our data show that human PDL cells in 3D cultures with the addition of CEMP1 has the potential to be used for the bioengineering reconstruction of periodontal tissues and cartilage since our results suggest that CEMP1 stimulates human PDL cells to differentiate towards different phenotypes.

Utilidad del control de la presión intraabdominal en el tratamiento de las eventraciones gigantes / Utility of intraabdominal pressure regulation in the treatment of giant eventrations

En las eventraciones gigantes la reintegración quirúrgica de las vísceras, posible bajo curarización, provoca aumentos de la presión intraabdominal que afectan a la evolución postoperatoria de estos pacientes. Valoramos la utilidad del control de la presión intravesical como guía durante el cierre de la pared abdominal para prevenir la aparición de hipertensión abdominal, así como durante el postoperatorio, lo que permite reconocer su presencia y establecer de forma temprana un tratamiento adecuado. (AU)