Osteogenic molecules for clinical applications: improving the BMP-collagen system.

Among the osteogenic growth factors used for bone tissue engineering, bone morphogenetic proteins (BMPs) are the most extensively studied for use in orthopaedic surgery. BMP-2 and BMP-7 have been widely investigated for developing therapeutic strategies and are the only two approved for use in several clinical applications. Due to the chemical and biological characteristics of these molecules, their authorised uses are always in combination with a carrier based on collagen type I. Although the use of these growth factors is considered safe in the short term, the very high doses needed to obtain significant osteoinduction make these treatments expensive and their long-term safety uncertain, since they are highly pleiotropic and have the capacity to induce ectopic ossification in the surrounding tissues. Therefore it is necessary to improve the currently used BMP-collagen system in terms of efficiency, biosecurity and costs. There are several strategies to increase the clinical effectiveness of these treatments. In this review we summarize the most promising results and our related work focused on this field through two different approaches: i) the development of recombinant BMPs with additional features, and ii) complementing these systems with other growth factors or molecules to enhance or accelerate osteogenesis.

Basic fibroblast growth factor enhances the osteogenic differentiation induced by bone morphogenetic protein-6 in vitro and in vivo.

Some members of the bone morphogenetic protein subfamily (BMP-2 and -7) are currently used in orthopedic surgery for several applications. Although their use is considered safe at short term, the high doses of growth factors needed make these treatments expensive and their safety uncertain at long term. BMP-6 has been much less studied than BMP-2 and -7, but some authors suggest that this BMP might have a stronger osteogenic activity than the previously mentioned. Having in mind that angiogenesis plays a well-known role during bone formation, the aim of this work was to study the effect of combining BMP-6 with bFGF on both the growth and differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs), as well as on in vivo osteogenesis. We demonstrate that a low dose of bFGF enhances the osteogenic differentiation of MSCs induced by BMP-6 in vitro. Furthermore, we also demonstrate that bone formation in vivo induced by BMP-6 can be accelerated and enhanced by adding a low dose of bFGF, what might suggest a synergic effect between these growth factors on in vivo osteogenesis.

Secretory Profile of Adipose-Tissue-Derived Mesenchymal Stem Cells from Cats with Calicivirus-Positive Severe Chronic Gingivostomatitis.

The feline calicivirus (FCV) causes infections in cats all over the world and seems to be related to a broad variety of clinical presentations, such as feline chronic gingivostomatitis (FCGS), a severe oral pathology in cats. Although its etiopathogeny is largely unknown, FCV infection is likely to be a main predisposing factor for developing this pathology. During recent years, new strategies for treating FCGS have been proposed, based on the use of mesenchymal stem cells (MSC) and their regenerative and immunomodulatory properties. The main mechanism of action of MSC seems to be paracrine, due to the secretion of many biomolecules with different biological functions (secretome). Currently, several pathologies in humans have been shown to be related to functional alterations of the patient's MSCs. However, the possible roles that altered MSCs might have in different diseases, including virus-mediated diseases, remain unknown. We have recently demonstrated that the exosomes produced by the adipose-tissue-derived MSCs (fAd-MSCs) from cats suffering from FCV-positive severe and refractory FCGS showed altered protein contents. Based on these findings, the goal of this work was to analyze the proteomic profile of the secretome produced by feline adipose-tissue-derived MSCs (fAd-MSCs) from FCV-positive patients with FCGS, in order to identify differences between them and to increase our knowledge of the etiopathogenesis of this disease. We used high-resolution mass spectrometry and functional enrichment analysis with Gene Ontology to compare the secretomes produced by the fAd-MSCs of healthy and calicivirus-positive FCGS cats. We found that the fAd-MSCs from cats with FCGS had an increased expression of pro-inflammatory cytokines and an altered proteomic profile compared to the secretome produced by cells from healthy cats. These findings help us gain insight on the roles of MSCs and their possible relation to FCGS, and may be useful for selecting specific biomarkers and for identifying new therapeutic targets.

Sponge-like processed D-periodic self-assembled atelocollagen supports bone formation in vivo.

Fibrous biopolymeric collagen extracted from animal tissues has been widely used for fabricating matrices for bone tissue engineering (BTE). However, animal extracted collagens can trigger immune reactions when implanted in vivo and the presence of native crosslinks leads to batch-to-batch variability. Atelocollagen, a monomeric form of collagen, is free of telopeptides, which are mainly responsible for the immunogenicity of collagen, and can self-assemble in vitro to obtain fibrils with the characteristic D-periodic staining pattern of native collagen. However, atelocollagen-based biomaterials have not extensively been studied and, hence, their suitability for BTE remains relatively unexplored. Besides, to stabilize collagen biomaterials, chemical and physical crosslinking are used, although chemical agents are cytotoxic while the physical methods yield a less effective crosslinking. A combination of physical and chemical crosslinking is a suitable alternative that has rarely been tested in BTE programs. In this work, a sponge-like biomaterial (DCol-S) was processed from D-periodic self-assembled atelocollagen and its stabilization was studied using the combination of a dehydrothermal treatment (DHT) and minimal glutaraldehyde (GTA) exposition crosslinking, to increase the resistance to degradation of the scaffold without a major effect on the biomaterial structure. The microstructural features of the final sponges were characterised and compared to a commercial biomaterial processed from native bovine collagen (Helistat®, Integra Lifesciences, NJ, USA), demonstrating that a D-periodic nanostructure was obtained and maintained after processing of the sponges. MC3T3-E1 preosteoblast adhesion, proliferation and differentiation assays in vitro showed that DCol-S is biocompatible. Furthermore, intramuscular implantation of the biomaterials loaded with rhBMP-2 revealed that the double-crosslinked sponges were able to support ectopic bone formation, while sponges stabilised only with the DHT treatment were not. Altogether, these findings show that atelocollagen-based sponges stabilised with a DHT treatment followed by a mild GTA crosslinking are a suitable alternative to polymeric extracted collagen for BTE applications.

Collagen Type I Biomaterials as Scaffolds for Bone Tissue Engineering.

Collagen type I is the main organic constituent of the bone extracellular matrix and has been used for decades as scaffolding material in bone tissue engineering approaches when autografts are not feasible. Polymeric collagen can be easily isolated from various animal sources and can be processed in a great number of ways to manufacture biomaterials in the form of sponges, particles, or hydrogels, among others, for different applications. Despite its great biocompatibility and osteoconductivity, collagen type I also has some drawbacks, such as its high biodegradability, low mechanical strength, and lack of osteoinductive activity. Therefore, many attempts have been made to improve the collagen type I-based implants for bone tissue engineering. This review aims to summarize the current status of collagen type I as a biomaterial for bone tissue engineering, as well as to highlight some of the main efforts that have been made recently towards designing and producing collagen implants to improve bone regeneration.

The Effect of Pore Directionality of Collagen Scaffolds on Cell Differentiation and In Vivo Osteogenesis.

Although many bone substitutes have been designed and produced, the development of bone tissue engineering products that mimic the microstructural characteristics of native bone remains challenging. It has been shown that pore orientation within collagen scaffolds influences bone matrix formation by the endochondral route. In addition, that the unidirectional orientation of the scaffolds can limit the growth of blood vessels. However, a comparison between the amount of bone that can be formed in scaffolds with different pore orientations in addition to analyzing the effect of loading osteogenic and proangiogenic factors is still required. In this work we fabricated uni- and multidirectional collagen sponges and evaluated their microstructural, physicochemical, mechanical and biological characteristics. Although the porosity and average pore size of the uni- and multidirectional scaffolds was similar (94.5% vs. 97.1% and 260 µm vs. 269 µm, respectively) the unidirectional sponges had a higher tensile strength, Young's modulus and capacity to uptake liquids than the multidirectional ones (0.271 MPa vs. 0.478 MPa, 9.623 MPa vs. 3.426 MPa and 8000% mass gain vs. 4000%, respectively). Culturing of rat bone marrow mesenchymal stem cells demonstrated that these scaffolds support cell growth and osteoblastic differentiation in the presence of BMP-2 in vitro, although the pore orientation somehow affected cell attachment and differentiation. The evaluation of the ability of the scaffolds to support bone growth when loaded with BMP-2 or BMP-2 + VEGF in an ectopic rat model showed that they both supported bone formation. Histological analysis and quantification of mineralized matrix revealed that the pore orientation of the collagen scaffolds influenced the osteogenic process.

Altered Proteomic Profile of Adipose Tissue-Derived Mesenchymal Stem Cell Exosomes from Cats with Severe Chronic Gingivostomatitis.

Feline chronic gingivostomatitis (FCGS) is a pathology with a complicated therapeutic approach and with a prevalence between 0.7 and 12%. Although the etiology of the disease is diverse, feline calicivirus infection is known to be a predisposing factor. To date, the available treatment helps in controlling the disease, but cannot always provide a cure, which leads to a high percentage of refractory animals. Mesenchymal stem cells (MSCs) play a pivotal role in the homeostasis and reparation of different tissues and have the ability to modulate the immune system responses. This ability is, in part, due to the capacity of exosomes to play a part in intercellular cell communication. However, the precise role of MSC-derived exosomes and their alterations in immunocompromised pathologies remains unknown, especially in veterinary patients. The goal of this work was to analyze the proteomic profile of feline adipose tissue-derived MSCs (fAd-MSCs) from calicivirus-positive FCGS patients, and to detect possible modifications of the exosomal cargo, to gain better knowledge of the disease's etiopathogenesis. Using high-resolution mass spectrometry and functional enrichment analysis with Gene Ontology, exosomes isolated from the fAd-MSCs of five healthy cats and five calicivirus-positive FCGS patients, were pooled and compared. The results showed that the fAd-MSCs from cats suffering from FCGS not only had a higher exosome production, but also their exosomes showed significant alterations in their proteomic profile. Eight proteins were exclusively found in the exosomes from the FCGS group, and five proteins could only be found in the exosomes from the healthy cats. When comparing the exosomal cargo between the two groups, significant upregulation of 17 and downregulation of 13 proteins were detected in the FCGS group compared to the control group. These findings shed light on new perspectives on the roles of MSCs and their relation to this disease, which may help in identifying new therapeutic targets and selecting specific biomarkers.

The subcommissural organ and the development of the posterior commissure in chick embryos.

The subcommissural organ (SCO) is an ependymal differentiation located in the diencephalon under the posterior commissure (PC). SCO-spondin, a glycoprotein released by the SCO, belongs to the thrombospondin superfamily and shares molecular domains with axonal pathfinding molecules. Several lines of evidence suggest a relationship between the SCO and the development of the PC in the chick: (1) their close location to each other, (2) their differentiation at the same developmental stage in the chick, (3) the abnormal PC found in null mutants lacking an SCO and (4) the release by the SCO of SCO-spondin. By application of DiI crystals in the PC of chick embryos, we have identified the neurons that give rise to the PC. Labelling is confined to the magnocellular nucleus of the PC (MNPC). To gain insight into the role of the SCO in PC development, coculture experiments of explants of the MNPC region (MNPCr) from embryos at embryonic day 4 (E4) with SCO explants from E4 or E13 embryos have been performed and the neurite outgrowth from the MNPCr explants has been analysed. In the case of coculture of E4 MNPCr with E4 SCO, the number of neurites growing from the MNPCr is higher at the side facing the SCO. However, when E4 MNPCr and E13 SCO are cocultured, the neurites grow mostly at the side opposite to the SCO. These data suggest that, at early stages of development, the SCO releases some attractive or permissive molecule(s) for the growing of the PC, whereas at later stages, the SCO has a repulsive effect over neurites arising from MNPCr.

Insulin-like growth factor-1 (IGF-1) enhances the osteogenic activity of bone morphogenetic protein-6 (BMP-6) in vitro and in vivo, and together have a stronger osteogenic effect than when IGF-1 is combined with BMP-2.

Bone morphogenetic protein-2 (BMP-2) is widely used in orthopedic surgery and bone tissue engineering because of its strong osteogenic activity. However, BMP-2 treatments have several drawbacks and many groups are actively exploring alternatives. Since BMP-6 has been demonstrated to be more osteoinductive, its use, either alone or together with other growth factors, might be an interesting option. In this work, we have compared the effect of BMP-2, BMP-6, or insulin-like growth factor-1 (IGF-1), either alone or in combination. Murine preosteoblasts were treated with 15 nM IGF-1 and/or 6 nM BMP-2 or -6 and the expression of osteogenic marker genes, proliferation, and alkaline phosphatase (ALP) activity in vitro were analyzed. The results showed that IGF-1 greatly enhanced the BMP-induced osteogenic differentiation of these cells in general and that the ALP activity in the cultures was higher when the combination was made with BMP-6 than with BMP-2. Furthermore, we tested the osteogenic potential of these treatments in vivo by loading 25 pmoles of IGF-1 and/or 10 pmoles of BMP-2 or -6 onto absorbable collagen sponges and implanting them into an ectopic bone formation model in rats. This study revealed that only BMP-6 was able to induce bone formation at the used dose and that the addition of IGF-1 contributed to an increase of the mineralization in the implants. Hence, the combination of BMP-6 with IGF-1 might be a better alternative than BMP-2 for orthopedic surgery or bone tissue engineering approaches. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1867-1875, 2017.

Combining bone morphogenetic proteins-2 and -6 has additive effects on osteoblastic differentiation in vitro and accelerates bone formation in vivo.

While only two members of the bone morphogenetic protein subfamily (BMP-2 and -7) are approved to be used in combination with collagen type I in orthopaedic surgery, other BMPs are known to also be highly osteoinductive. Although all the osteogenic BMPs signal through Smad-1/-5/-8 phosphorylation, they show different preferences for the available BMP receptors. In this work we studied the effect of combining two osteogenic BMPs (-2 and -6), which belong to different groups within the subfamily and have different affinities to the existing BMP receptors. Both the growth and in vitro differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs) were studied, as well as in vivo ectopic bone formation when the BMPs were intramuscularly implanted in rats with collagen type I sponges as carriers. The results show that these two growth factors have additive effects on the osteoblastic differentiation of cells in vitro and that their combination might be helpful to accelerate in vivo osteogenesis while reducing the amount of each individual BMP used.

Peptides for bone tissue engineering.

Molecular signals in the form of growth factors are the main modulators of cell behavior. However, the use of growth factors in tissue engineering has several drawbacks, including their costs, difficult production, immunogenicity and short half-life. Furthermore, many of them are pleiotropic and, since a single growth factor can have different active domains, their effect is not always fully controllable. A very interesting alternative that has recently emerged is the use of biomimetic peptides. Sequences derived from the active domains of soluble or extracellular matrix proteins can be used to functionalize the biomaterials used as scaffolds for new tissue growth to either direct the attachment of cells or to be released as soluble ligands. Since these short peptides can be easily designed and cost-effectively synthesized in vitro, their use has opened up a world of new opportunities to obtain cheaper and more effective implants for regenerative medicine strategies. In this extensive review we will go through many of the most important peptides with potential interest for bone tissue engineering, not limiting to those that only mediate cell adhesion or induce the osteogenic differentiation of progenitor cells, but also focusing on those that direct angiogenesis because of its close relation with bone formation.

A collagen-targeted biomimetic RGD peptide to promote osteogenesis.

Osteogenesis is a complex, multifactorial process in which many different signals interact. The bone morphogenetic proteins (BMPs) are the most potent inducers of osteoblastic differentiation, although very high doses of BMPs in combination with collagen type I formulations have to be used for clinical applications. Although integrin-binding arginine-glycine-aspartic acid (RGD) biomimetic peptides have shown some promising abilities to promote the attachment of cells to biomaterials and to direct their differentiation, the linking of these peptides to collagen sponges usually implies chemical manipulation steps. In this study, we describe the design and characterization of a synthetic collagen-targeted RGD biomimetic (CBD-RGD) peptide formed from a collagen-binding domain derived from the von Willebrand factor and the integrin-binding RGD sequence. This peptide was demonstrated to bind to absorbable collagen type I sponges (ACSs) without performing any chemical linking, and to induce the differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells. Furthermore, in vivo experiments showed that ACSs functionalized with CBD-RGD and loaded with a subfunctional dose of BMP-2-formed ectopic bone in rats, while nonfunctionalized sponges loaded with the same amount of BMP-2 did not. These results indicate that the combination of this biomimetic peptide with the currently used collagen+BMP system might be a promising approach to improve osteogenesis and to reduce the doses of BMPs needed in clinical orthopedics.

The subcommissural organ and the development of the posterior commissure.

Growing axons navigate through the developing brain by means of axon guidance molecules. Intermediate targets producing such signal molecules are used as guideposts to find distal targets. Glial, and sometimes neuronal, midline structures represent intermediate targets when axons cross the midline to reach the contralateral hemisphere. The subcommissural organ (SCO), a specialized neuroepithelium located at the dorsal midline underneath the posterior commissure, releases SCO-spondin, a large glycoprotein belonging to the thrombospondin superfamily that shares molecular domains with axonal pathfinding molecules. Several evidences suggest that the SCO could be involved in the development of the PC. First, both structures display a close spatiotemporal relationship. Second, certain mutants lacking an SCO present an abnormal PC. Third, some axonal guidance molecules are expressed by SCO cells. Finally, SCO cells, the Reissner's fiber (the aggregated form of SCO-spondin), or synthetic peptides from SCO-spondin affect the neurite outgrowth or neuronal aggregation in vitro.

The effect of an rhBMP-2 absorbable collagen sponge-targeted system on bone formation in vivo.

Reparation of bone defects remains a major clinical and economic concern, with more than 3 million bone grafts performed annually only in the United States and the EU. The search for alternatives to autologous bone grafting led to the approval by the FDA of an absorbable collagen carrier combined with rhBMP-2 for the treatment of certain bone diseases and fractures. The present work is focused on the production of a collagen-targeted rhBMP-2 based system to improve bone formation. We produced a modified rhBMP-2 with only an additional collagen-binding decapeptide derived from the von Willebrand factor and tested its affinity to collagen and its ability to induce ectopic bone formation in vivo when implanted in combination with absorbable collagen sponges or hydroxyapatite. The results showed not only that the rhBMP2-CBD had an increased affinity to collagen, but also that this binding was very stable during a prolonged period of time. In vivo experiments demonstrated that this rhBMP2-CBD maintained its osteoinductive activity, being capable of inducing new bone formation even at lower concentrations than native rhBMP-2. These results indicate that the combination of the fusion protein with absorbable collagen may be a suitable and safer alternative to rhBMP-2 for bone repair purposes.

Osteogenic molecules for clinical applications: improving the BMP-collagen system

Among the osteogenic growth factors used for bone tissue engineering, bone morphogenetic proteins (BMPs) are the most extensively studied for use in orthopaedic surgery. BMP-2 and BMP-7 have been widely investigated for developing therapeutic strategies and are the only two approved for use in several clinical applications. Due to the chemical and biological characteristics of these molecules, their authorised uses are always in combination with a carrier based on collagen type I. Although the use of these growth factors is considered safe in the short term, the very high doses needed to obtain significant osteoinduction make these treatments expensive and their long-term safety uncertain, since they are highly pleiotropic and have the capacity to induce ectopic ossification in the surrounding tissues. Therefore it is necessary to improve the currently used BMP-collagen system in terms of efficiency, biosecurity and costs. There are several strategies to increase the clinical effectiveness of these treatments. In this review we summarize the most promising results and our related work focused on this field through two different approaches: i) the development of recombinant BMPs with additional features, and ii) complementing these systems with other growth factors or molecules to enhance or accelerate osteogenesis.