Combining canine mesenchymal stromal cells and hyaluronic acid for cartilage repair.

Cell therapy and tissue engineering have been intensively researched for repair of articular cartilage. In this study, we investigated the chondrogenic potential of canine adipose-derived mesenchymal stromal cells (ASCs) combined to high molecular weight hyaluronic acid (HA) in vitro, and their therapeutic effect in dogs with chronic osteoarthritis (OA) associated with bilateral hip dysplasia. Canine ASCs were characterized after conventional 2D culture or 3D culture in HA, showing adequate immunophenotype, proliferation and trilineage differentiation, as well as chondrogenesis after cultivation in HA. ASC/HA constructs were used to treat 12 dogs with OA, sequentially assigned to control, ASC and ASC/HA groups. Animals were examined for clinical, orthopedic and radiological parameters. Lameness at walk and pain on manipulation were reduced in the ASC group and mainly in the ASC/HA group. Range of motion and detection of crepitus on hip rotation and abduction improved similarly in all groups. For articular edema, muscle atrophy, Norberg angle values and radiographic analyses, there were no variations throughout the period. These results indicate that ASC/HA constructs are safe and may be an effective therapeutic tool in treating canine chronic osteoarthritis, which should be confirmed with larger studies and additional clinical parameters.

Coronary corium, a new source of equine mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have attracted great attention for therapeutic applications. Since cells derived from different tissues have different properties, using the right tissue source may impact their efficiency in regenerative medicine. This study describes for the first time the isolation and characterization of MSCs derived from the equine coronary corium, which may be useful for treating diseases such as laminitis. Seven coronary corium samples were used for isolation of cells (ccMSCs). Adherent cells were characterized for morphology, immunophenotype, proliferation and differentiation potential, in vitro migration and colony-forming capacity. The cells displayed the characteristic fibroblastoid morphology, with population doubling time increasing until passage 7 and reaching a plateau in passage 10. Cells were negative for CD14 and CD45, and positive for CD73 and CD90. ccMSCs showed chondrogenic and osteogenic, but not adipogenic differentiation, and migrated with nearly total closing of the empty area in 48 h, in the scratch assay. The clonogenic potential was in average 18% to 23%. This study describes for the first time the establishment of mesenchymal stromal cell cultures from the equine coronary corium. The results are similar to MSCs isolated from many other equine tissues, except for restricted differentiation potential. As coronary corium stem cell regulation may contribute to the pathogenesis of equine chronic laminitis, the use of ccMSCs in cell therapy for this significantly debilitating disease should be further investigated.

Chondrogenic effect of liquid and gelled platelet lysate on canine adipose-derived mesenchymal stromal cells.

Osteoarthritis associated with hip dysplasia is one of the most common orthopedic abnormalities in dogs, with an incidence of up to 40% in some breeds. Tissue therapy of cartilage has received great attention, with use of mesenchymal stromal cells and different types of biomaterials. The present study aimed to evaluate the effect of platelet lysate (PL) on the proliferation and differentiation of canine adipose tissue-derived mesenchymal stromal cells (ASCs), in liquid culture or hydrogels. PL was prepared from blood collected from healthy dogs and submitted to freezing-thawing cycles, and hydrogel was formed with canine thrombin. The effect of PL on the proliferation and differentiation of canine ASCs was evaluated in liquid and hydrogel systems, with microscopy, quantification of dsDNA, histology and quantification of glycosaminoglycans. The addition of 5% or 10% PL to the culture medium induced a greater proliferation rate than the presence of 10% fetal bovine serum. The cultivation of ASCs in PL gel, with normal or chondrogenic medium, resulted in maintenance of proliferation level similar to the conventional 2D cultivation, and induction of chondrogenic differentiation, especially in the presence of the chondrogenesis induction medium.

Isolation and characterization of mesenchymal stem/stromal cells from Ctenomys minutus.

Mesenchymal stem/stromal cells (MSCs) are multipotent cells distributed in all tissues and characterized by adherence, morphology, immunophenotype and trilineage differentiation potential. The present study aimed to isolate and characterize adherent MSC-like populations from different tissues of Ctenomys minutus, a threatened wildlife rodent popularly known as tuco-tuco. Adherent cells were isolated from bone marrow, brain, liver, pancreas and adipose tissue of three adult animals collect in southern Brazil. Cultures showed typical morphology and proliferation potential. Adipose-derived MSCs showed trilineage potential. Cultures derived from adipose tissue, bone marrow and brain were immunophenotyped with negative results for CD31, CD44, CD45, CD106, and MHC class II, as well as strong positive results for CD29. Low fluorescence levels were seen for CD49d, CD90.2 and CD117. Cultures were negative for CD49e, except for brain-derived cultures that were weakly positive. CD11b was negative in adipose-derived MSCs, but positive in brain and bone marrow-derived cultures. The scratch assay showed high migration potential for pancreas and adipose tissue-derived cells. This study represents the first report of isolation and characterization of cultures having characteristics of MSCs from Ctenomys minutus. The collection of biological information for biobanks represents an important contribution to the creation of strategies for prevention of loss of genetic diversity.

Adipose-derived stem cells in veterinary medicine: characterization and therapeutic applications.

Mesenchymal stem cells, considered one of the most promising cell types for therapeutic applications due to their capacity to secrete regenerative bioactive molecules, are present in all tissues. Stem cells derived from the adipose tissue have been increasingly used for cell therapy in humans and animals, both as freshly isolated, stromal vascular fraction (SVF) cells, or as cultivated adipose-derived stem cells (ASCs). ASCs have been characterized in different animal species for proliferation, differentiation potential, immunophenotype, gene expression, and potential for tissue engineering. Whereas canine and equine ASCs are well studied, feline cells are still poorly known. Many companies around the world offer ASC therapy for dogs, cats, and horses, although in most countries these activities are not yet controlled by regulatory agencies. This is the first study to review the characterization and clinical use of SVF and ASCs in spontaneously occurring diseases in veterinary patients. Although a relatively large number of studies investigating ASC therapy in induced lesions are available in the literature, a surprisingly small number of reports describe ASC therapy for naturally affected dogs, cats, and horses. A total of seven studies were found with dogs, only two studies in cats, and four in horses. Taken as a whole, the results do not allow a conclusion on the effect of this therapy, due to the generally small number of patients included, diversity of cell populations used, and lack of adequate controls. Further controlled studies are clearly needed to establish the real potential of ASC in veterinary medicine.

Acupoint injection of autologous stromal vascular fraction and allogeneic adipose-derived stem cells to treat hip dysplasia in dogs.

Stem cells isolated from adipose tissue show great therapeutic potential in veterinary medicine, but some points such as the use of fresh or cultured cells and route of administration need better knowledge. This study aimed to evaluate the effect of autologous stromal vascular fraction (SVF, n = 4) or allogeneic cultured adipose-derived stem cells (ASCs, n = 5) injected into acupuncture points in dogs with hip dysplasia and weak response to drug therapy. Canine ASCs have proliferation and differentiation potential similar to ASCs from other species. After the first week of treatment, clinical evaluation showed marked improvement compared with baseline results in all patients treated with autologous SVF and three of the dogs treated with allogeneic ASCs. On days 15 and 30, all dogs showed improvement in range of motion, lameness at trot, and pain on manipulation of the joints, except for one ASC-treated patient. Positive results were more clearly seen in the SVF-treated group. These results show that autologous SVF or allogeneic ASCs can be safely used in acupoint injection for treating hip dysplasia in dogs and represent an important therapeutic alternative for this type of pathology. Further studies are necessary to assess a possible advantage of SVF cells in treating joint diseases.

Isolation of adipose-derived stem cells: a comparison among different methods.

Adipose-derived stromal cells (ASCs) are usually isolated by digestion with collagenase. We have compared alternative methods to isolate ASCs in a more economically viable protocol. Nine protocols using red blood cells lysis buffer solution, trypsin, collagenase and centrifugation were compared; the isolation rate, cell viability, expansion rate, immunophenotype and differentiation in adipogenic and osteogenic lineages were analyzed. ASCs were isolated and successfully maintained by digestion with trypsin. Cells presented similar immunophenotypes, adipogenic differentiation and in vitro proliferation but an osteogenic differentiation capacity up to seven times higher than ASCs isolated by collagenase. This alternative protocol is thus efficient and more cost-effective than the commonly-used methods and may represent a promising protocol for obtaining ASCs for bone tissue engineering.