Rick Visser

Mesenchymal stem cells (MSCs) differentiate to cells that are part of tissues, such as bone and cartilage, both during ontogeny and for the repair/regeneration and renewal of tissues during postnatal life. Accordingly, MSCs can be a powerful tool for regenerative medicine and tissue engineering interventions in musculoskeletal deficiencies. MSCs found in the bone marrow and connective tissues are exposed to biochemical and biophysical factors that modulate their biological behavior. Given that biochemical factors have been widely studied, research on the effect of biophysical stimuli on the differentiation of MSCs is scarce. An overview of the impact of biophysical factors on MSCs osteogenic differentiation could shed light on some of the signals to consider when developing new cell therapies or tissue engineering products for the treatment of bone traumatic defects and diseases. The aim of this review is to provide an overview of the effect of biophysical stimuli on osteogenic differentiation. It focuses on reviewing what has been published on the effect of compression, tension, hydrostatic pressure, shear stress, electromagnetic fields, low-intensity ultrasound, and niche signaling – including topography and substrate stiffness, paracrine and juxtracrine regulation – on MSCs osteogenic differentiation. The knowledge gained from studying how MSCs respond to mechanical forces has the potential to drive the creation of more advanced treatments for conditions where bones are injured. These therapies could rely on either physical methods or a combination of physical and chemical approaches, offering new possibilities for improving bone health.

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.

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.

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.

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.

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.

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. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 178–185, 2016.

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.