Modulation of Canine Adipose-Derived Mesenchymal Stem/Medicinal Signalling Cells with Ascorbic Acid: Effect on Proliferation and Chondrogenic Differentiation on Standard Plastic and Silk Fibroin Surfaces.

Ascorbic acid (AA) plays a crucial role in both the proliferation and chondrogenic differentiation potential of mesenchymal stem/medicinal signalling cells (MSCs); these are both key aspects of their general therapeutic use and their increasing use in veterinary medicine. Current immunomodulatory therapies require efficient expansion of MSCs in the laboratory, while emerging tissue regeneration strategies, such as cartilage or bone repair, aim to use differentiated MSCs and modulate the expression of chondrogenic and hypertrophic markers. Our aim was to investigate whether the addition of AA to the growth medium enhances the proliferation of canine adipose-derived MSCs (cAMSCs) grown on standard plastic surfaces and whether it affects chondrogenic differentiation potential on silk fibroin (SF) films. We assessed cell viability with trypan blue and proliferation potential by calculating population doubling. Chondrogenic induction on SF films was assessed by Alcian blue staining and gene expression analysis of chondrogenic and hypertrophic genes. The results showed that growth medium with AA significantly enhanced the proliferation of cAMSCs without affecting cell viability and modulated the expression of chondrogenic and hypertrophic genes of cAMSCs grown on SF films. Our results suggest that AA may be used in growth medium for expansion of cAMSCs and, at the same time, provide the basis for future studies to investigate the role of AA and SF in chondrogenic differentiation of MSCs.

Articular Cartilage Regeneration in Veterinary Medicine.

Cartilage is an avascular tissue with a limited rate of oxygen and nutrient diffusion, resulting in its inability to heal spontaneously. Articular cartilage defects eventually lead to osteoarthritis (OA), the endpoint of progressive destruction of cartilage. In companion animals, OA is the most common joint disease, and many pain management and surgical attempts have been made to find an appropriate treatment. Pain management of OA is usually the first choice of OA therapy, which is often managed with nonsteroidal anti-inflammatory drugs (NSAIDs). To avoid known negative side effects of NSAIDs, other approaches are being considered, such as the use of anti-nerve growth factor monoclonal antibodies (anti-NGF mAB), hyaluronic acid (HA), platelet-rich plasma (PRP), and mesenchymal stem cells (MSCs). The latter is increasingly being recognized as effective in reducing or even eliminating pain and lameness associated with OA. However, the in vivo mechanisms of MSC action do not relate to their differentiation potential, but rather to their immunomodulatory functions. Achieving actual regeneration of cartilage to prevent OA from developing or even revert already existing OA condition has not yet been achieved. Several techniques have been tried to overcome cartilage's inability to regenerate, from osteochondral transplantation, autologous chondrocyte implantation (ACI), and matrix-induced ACI (MACI). Combinatory use of MSCs unique features and biomaterials is also being investigated with the aim to as much as possible recapitulate the native microenvironment of the cartilage, yet so far none of the methods have produced reliable and truly effective results. Although OA, for now, remains an incurable disease, novel techniques are being developed, rendering hope for the future accomplishment of actual cartilage regeneration. The aim of this chapter is firstly to summarize known and developing pain management options for OA, secondly to present surgical attempts to regenerate articular cartilage, and finally to present the attempts to improve existing regenerative treatment options using mesenchymal stem cells, with the vision for the possible use of developing strategies in veterinary medicine.

Treatment of cranial cruciate ligament injuries in dogs using a combination of tibial tuberosity advancement procedure and autologous mesenchymal stem cells/multipotent mesenchymal stromal cells - A pilot study.

In the present pilot study, we evaluated different supplemental therapies using autologous multipotent mesenchymal stromal cells (MMSCs) for the treatment of cranial cruciate ligament defects in dogs. We used tibial tuberosity advancement (TTA) and augmented it by supportive therapy with MMSCs in three patient groups. In the first patient group, the dogs were injected with MMSCs directly into the treated stifle one month after surgery. In the second group, MMSCs were delivered in a silk fibroin scaffold which was placed in the osteotomy gap during surgery. In the third group, MMSCs were first mixed with bone tissue and blood from the patient and delivered into the osteotomy gap during surgery. In the control group, patients underwent the TTA procedure but did not receive MMSC treatment. In the group of patients who received cells in the silk fibroin scaffold during surgery, the osteotomy gap did not heal, presumably due to the low absorption of silk fibroin. Patients who received MMSCs mixed with bone tissue and blood during surgery into the osteotomy gap recovered clinically faster and had better healing of the osteotomy gap than dogs from the other two treated groups and from the control group, as assessed by clinical examination and quantification of radiographs. In conclusion, dogs that received stem cells directly into the osteotomy gap (Group 3) recovered faster compared to dogs from Groups 1 (MMSCs injected into the joint one month after surgery), 2 (cells implanted into the osteotomy gap in a silk fibroin scaffold), and the control group that did not receive additional MMSCs treatment.

Stem Cells in Veterinary Medicine-Current State and Treatment Options.

Regenerative medicine is a branch of medicine that develops methods to grow, repair, or replace damaged or diseased cells, organs or tissues. It has gained significant momentum in recent years. Stem cells are undifferentiated cells with the capability to self-renew and differentiate into tissue cells with specialized functions. Stem cell therapies are therefore used to overcome the body's inability to regenerate damaged tissues and metabolic processes after acute or chronic insult. The concept of stem cell therapy was first introduced in 1991 by Caplan, who proposed that massive differentiation of cells into the desired tissue could be achieved by isolation, cultivation, and expansion of stem cells in in vitro conditions. Among different stem cell types, mesenchymal stem cells (MSC) currently seem to be the most suitable for therapeutic purposes, based on their simple isolation and culturing techniques, and lack of ethical issues regarding their usage. Because of their remarkable immunomodulatory abilities, MSCs are increasingly gaining recognition in veterinary medicine. Developments are primarily driven by the limitations of current treatment options for various medical problems in different animal species. MSCs represent a possible therapeutic option for many animal diseases, such as orthopedic, orodental and digestive tract diseases, liver, renal, cardiac, respiratory, neuromuscular, dermal, olfactory, and reproductive system diseases. Although we are progressively gaining an understanding of MSC behavior and their mechanisms of action, some of the issues considering their use for therapy are yet to be resolved. The aim of this review is first to summarize the current knowledge and stress out major issues in stem cell based therapies in veterinary medicine and, secondly, to present results of clinical usage of stem cells in veterinary patients.

Comparison of Canine and Feline Adipose-Derived Mesenchymal Stem Cells/Medicinal Signaling Cells With Regard to Cell Surface Marker Expression, Viability, Proliferation, and Differentiation Potential.

Remarkable immunomodulatory abilities of mesenchymal stem cells, also called multipotent mesenchymal stromal cells or medicinal signaling cells (MSCs), have entailed significant advances in veterinary regenerative medicine in recent years. Despite positive outcomes from MSC therapies in various diseases in dogs and cats, differences in MSC characteristics between small animal veterinary patients are not well-known. We performed a comparative study of cells' surface marker expression, viability, proliferation, and differentiation capacity of adipose-derived MSCs (ADMSCs) from dogs and domestic cats. The same growth media and methods were used to isolate, characterize, and culture canine and feline ADMSCs. Adipose tissue was collected from 11 dogs and 8 cats of both sexes. The expression of surface markers CD44, CD90, and CD34 was detected by flow cytometry. Viability at passage 3 was measured with the hemocytometer and compared to the viability measured by flow cytometry after 1 day of handling. The proliferation potential of MSCs was measured by calculating cell doubling and cell doubling time from second to eighth passage. Differentiation potential was determined at early and late passages by inducing cells toward adipogenic, osteogenic, and chondrogenic differentiation using commercial media. Our study shows that the percentage of CD44+CD90+ and CD34-/- cells is higher in cells from dogs than in cells from cats. The viability of cells measured by two different methods at passage 3 differed between the species, and finally, canine ADMSCs possess greater proliferation and differentiation potential in comparison to the feline ADMSCs.

Silk fibroin induces chondrogenic differentiation of canine adipose-derived multipotent mesenchymal stromal cells/mesenchymal stem cells.

Under appropriate culture conditions, mesenchymal stem cells (MSC), also called more properly multipotent mesenchymal stromal cells (MMSC), can be induced toward differentiation into different cell lineages. In order to guide stem cell fate within an environment resembling the stem cell niche, different biomaterials are being developed. In the present study, we used silk fibroin (SF) as a biomaterial supporting the growth of MMSC and studied its effect on chondrogenesis of canine adipose-derived MMSC (cADMMSC). Adipose tissue was collected from nine privately owned dogs. MMSC were cultured on SF films and SF scaffolds in a standard cell culture medium. Cell morphology was evaluated by scanning electron microscopy (SEM). Chondrogenic differentiation was evaluated by alcian blue staining and mRNA expression of collagen type 1, collagen type 2, Sox9, and Aggrecan genes. cADMMSC cultured on SF films and SF scaffolds stained positive using alcian blue. SEM images revealed nodule-like structures with matrix vesicles and fibers resembling chondrogenic nodules. Gene expression of chondrogenic markers Sox9 and Aggrecan were statistically significantly upregulated in cADMMSC cultured on SF films in comparison to negative control cADMMSC. This result suggests that chondrogenesis of cADMMSC could occur when cells were grown on SF films in a standard cell culture medium without specific culture conditions, which were previously considered necessary for induction of chondrogenic differentiation.