Francisco José Vázquez

Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue.

Bone marrow and adipose tissue are the two main sources of mesenchymal stem cell (MSC). The aim of this work was to analyse the immunophenotype of 7 surface markers and the expression of a panel of 13 genes coding for cell surface markers in equine bone marrow and adipose tissue-derived MSCs obtained from 9 horses at third passage. The tri-lineage differentiation was confirmed by specific staining. Equine MSCs from both sources were positive for the MSC markers CD29 and CD90, while were negative for CD44, CD73, CD105, CD45 and CD34. The gene expression of these molecules was also evaluated by reverse transcriptase real-time quantitative PCR along with the expression of 5 other MSC markers. Both populations of cells expressed CD13, CD29, CD44, CD49d, CD73, CD90, CD105, CD106, CD146 and CD166 transcripts. Significant differences in gene expression levels between BM- and AT-MSCs were observed for CD44, CD90, CD29 and CD34. Both cell types were negative for CD45 and CD31. The surface antigens tested revealed a similar phenotypic profile between horse and human MSCs, although specific differences in some surface antigens were noticed.

Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue.

BackgroundMesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2.ResultsAt the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state.ConclusionsHypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.

Expansion under hypoxic conditions enhances the chondrogenic potential of equine bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely used in regenerative medicine in horses. Most of the molecular characterisations of BM-MSCs have been made at 20% O(2), a higher oxygen level than the one surrounding the cells inside the bone marrow. The present work compares the lifespan and the tri-lineage potential of equine BM-MSCs expanded in normoxia (20% O(2)) and hypoxia (5% O(2)). No significant differences were found in long-term cultures for osteogenesis and adipogenesis between normoxic and hypoxic expanded BM-MSCs. An up-regulation of the chondrogenesis-related genes (COL2A1, ACAN, LUM, BGL, and COMP) and an increase of the extracellular sulphated glycosaminoglycan content were found in cells that were expanded under hypoxia. These results suggest that the expansion of BM-MSCs in hypoxic conditions enhances chondrogenesis in equine BM-MSCs.

Porous orthopedic steel implant as an antibiotic eluting device: Prevention of post-surgical infection on an ovine model

Traumatology and orthopedic surgery can benefit from the use of efficient local antibiotic-eluting systems to avoid bacterial contamination of implanted materials. In this work a new percutaneous porous-wall hollow implant was successfully used as a local antibiotic-eluting device both in vitro and in vivo. The implant is a macroporous 316 L stainless steel filter tube with a nominal filtration cut-off size of 200 nm with one open end which was used to load the synthetic antibiotic linezolid and an opposite blind end. The antibiotic release kinetics from the device on a simulated biological fluid under in vitro conditions demonstrated an increased concentration during the first five days that subsequently was sustained for at least seven days, showing a kinetic close to a zero order release. Antibiotic-loaded implants were placed in the tibia of four sheep which were trans-surgically experimentally infected with a biofilm forming strain of Staphylococcus aureus. After 7 and 9 days post infection, sheep did not show any evidence of infection as demonstrated by clinical, pathological and microbiological findings. These results demonstrate the capability of such an antibiotic-loaded implant to prevent infection in orthopedic devices in vivo. Further research is needed to assess its possible use in traumatology and orthopedic surgery.

Expression of genes involved in immune response and in vitro immunosuppressive effect of equine MSCs.

The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation of PBMCs. The effect of the dose and source of MSCs was evaluated in cocultures of 10(5) PBMCs with different ratios of AT- and BM-MSCs (1:1, 1:10, 1:20 and 1:50). Proliferation rates of the PBMCs were evaluated using BrdU ELISA colorimetric assay. PHA stimulated equine PBMCs reached their peak of growth after 3 days of culture. The immunoassay showed a decrease of the PBMCs growth at high ratio cocultures (1:1 and 1:10). Equine BM-MSCs and AT-MSCs demonstrated an ability to suppress the proliferation of stimulated PBMCs. Although MSCs derived from both sources displayed immunosuppressive effects, AT-MSCs were slightly more potent than BM-MSCs. In addition, the expression of 26 genes coding for different molecules implicated in the immune response was analyzed in cocultures of BM-MSCs and PHA stimulated PBMSCs by reverse transcriptase real time quantitative PCR (RT-qPCR). An upregulation in genes associated with the production of interleukins and cytokines such as TNF-α and TGF-β1 was observed except for IFN-γ whose expression significantly decreased. The variations of interleukins and cytokine receptors showed no clear patterns. COX-1 and COX-2 showed similar expression patterns while INOs expression significantly decreased in the two cell types present in the coculture. Cyclin D2 and IDO-1 showed an increased expression and CD90, ITG-β1 and CD44 expression decreased significantly in BM-MSCs cocultured with PHA stimulated PBMCs. On the contrary, CD6 and VCAM1 expression increased in these cells. With regard to the expression of the five genes involved in antigen presentation, an upregulation was observed in both cocultured MSCs and stimulated PBMCs. This study contributes to the knowledge of the immunoregulatory properties of equine MSCs, which are notably important for the treatment of inflammation processes, such as tendinitis and osteoarthritis.

Transmission of sheep-bovine spongiform encephalopathy to pigs

Experimental transmission of the bovine spongiform encephalopathy (BSE) agent has been successfully reported in pigs inoculated via three simultaneous distinct routes (intracerebral, intraperitoneal and intravenous). Sheep derived BSE (Sh-BSE) is transmitted more efficiently than the original cattle-BSE isolate in a transgenic mouse model expressing porcine prion protein. However, the neuropathology and distribution of Sh-BSE in pigs as natural hosts, and susceptibility to this agent, is unknown. In the present study, seven pigs were intracerebrally inoculated with Sh-BSE prions. One pig was euthanized for analysis in the preclinical disease stage. The remaining six pigs developed neurological signs and histopathology revealed severe spongiform changes accompanied by astrogliosis and microgliosis throughout the central nervous system. Intracellular and neuropil-associated pathological prion protein (PrPSc) deposition was consistently observed in different brain sections and corroborated by Western blot. PrPSc was detected by immunohistochemistry and enzyme immunoassay in the following tissues in at least one animal: lymphoid tissues, peripheral nerves, gastrointestinal tract, skeletal muscle, adrenal gland and pancreas. PrPSc deposition was revealed by immunohistochemistry alone in the retina, optic nerve and kidney. These results demonstrate the efficient transmission of Sh-BSE in pigs and show for the first time that in this species propagation of bovine PrPSc in a wide range of peripheral tissues is possible. These results provide important insight into the distribution and detection of prions in non-ruminant animals.

Comparison of autologous bone marrow and adipose tissue derived mesenchymal stem cells, and platelet rich plasma, for treating surgically induced lesions of the equine superficial digital flexor tendon

Several therapies have been investigated for equine tendinopathies, but satisfactory long term results have not been achieved consistently and a better understanding of the healing mechanism elicited by regenerative therapies is needed. The aim of this study was to assess the separate effects of autologous bone marrow (BM) and adipose tissue (AT) derived mesenchymal stem cells (MSCs), and platelet rich plasma (PRP), for treating lesions induced in the superficial digital flexor tendon (SDFT) of horses. Lesions were created surgically in both SDFTs of the forelimbs of 12 horses and were treated with BM-MSCs (six tendons), AT-MSCs (six tendons) or PRP (six tendons). The remaining six tendons received lactated Ringers solution as control. Serial ultrasound assessment was performed prior to treatment and at 2, 6, 10, 20 and 45 weeks post-treatment. At 45 weeks, histopathology and gene expression analyses were performed. At week 6, the ultrasound echogenicity score in tendons treated with BM-MSCs suggested earlier improvement, whilst all treatment groups reached the same level at week 10, which was superior to the control group. Collagen orientation scores on histological examination suggested a better outcome in treated tendons. Gene expression was indicative of better tissue regeneration after all treatments, especially for BM-MSCs, as suggested by upregulation of collagen type I, decorin, tenascin and matrix metalloproteinase III mRNA. Considering all findings, a clear beneficial effect was elicited by all treatments compared with the control group. Although differences between treatments were relatively small, BM-MSCs resulted in a better outcome than PRP and AT-MSCs.

Acute phase protein haptoglobin as inflammatory marker in serum and synovial fluid in an equine model of arthritis.

Acute phase proteins are useful inflammatory markers in horses. Haptoglobin (Hp) serum level is increased in horses undergoing different inflammatory processes, including arthritis. However, Hp concentration has not been assessed in inflammatory synovial fluid (SF). The aim of the present study was to investigate the Hp response in serum and SF in horses undergoing experimentally induced arthritis. For this purpose, serum and SF samples were collected from 12 animals before amphotericin B-induced arthritis was created (T0, healthy) and 15days after the lesion induction (T1, joint inflammation) and Hp was determined by single radial immunodiffusion. The Hp increase between T0 and T1 was significant in both serum and SF, and serum Hp concentration at T0 was significantly higher than in SF, but significant differences were not found at T1, indicating a higher Hp increase in SF. A significant positive correlation for Hp concentration between serum and SF samples was found. These results highlight the potential usefulness of Hp as inflammatory marker in horses, showing for the first time the increase of Hp in SF from joint inflammation in the horse.

Inflammation affects the viability and plasticity of equine mesenchymal stem cells: possible implications in intra-articular treatments

Mesenchymal stem cells (MSCs) are gaining relevance for treating equine joint injuries because of their ability to limit inflammation and stimulate regeneration. Because inflammation activates MSC immunoregulatory function, proinflammatory priming could improve MSC efficacy. However, inflammatory molecules present in synovial fluid or added to the culture medium might have deleterious effects on MSCs. Therefore, this study was conducted to investigate the effects of inflammatory synovial fluid and proinflammatory cytokines priming on viability and plasticity of equine MSCs. Equine bone marrow derived MSCs (eBM-MSCs) from three animals were cultured for 72 h in media supplemented with: 20% inflammatory synovial fluid (SF); 50 ng/mL IFN-γ and TNF-α (CK50); and 20 ng/mL IFN-γ and TNF-α (CK20). Proliferation assay and expression of proliferation and apoptosis-related genes showed that SF exposed-eBM-MSCs maintained their viability, whereas the viability of CK primed-eBM-MSCs was significantly impaired. Tri-lineage differentiation assay revealed that exposure to inflammatory synovial fluid did not alter eBM-MSCs differentiation potential; however, eBM-MSCs primed with cytokines did not display osteogenic, adipogenic or chondrogenic phenotype. The inflammatory synovial environment is well tolerated by eBM-MSCs, whereas cytokine priming negatively affects the viability and differentiation abilities of eBM-MSCs, which might limit their in vivo efficacy.

Antibiotic-eluting orthopedic device to prevent early implant associated infections: Efficacy, biocompatibility and biodistribution studies in an ovine model: Infection Prevention Using Drug Eluting Orthopedic Implants

Infection of orthopedic devices is a major complication in the postsurgical period generating important health issues and economic consequences. Prevention strategies could be based on local release of antibiotics from the orthopedic device itself to avoid adhesion and growth of bacteria. The purpose of this work is to demonstrate the efficiency to prevent these infections by a cefazolin-eluting, perforated stainless steel implant in an in vivo ovine model. The device was placed in the tibia of sheep, one group receiving cefazolin-loaded implants whereas the control group received empty implants. All implants were experimentally infected by direct inoculation of Staphylococcus aureus ATCC 6538. In vitro cytotoxicological studies were also performed to check the effect of antibiotic on cell viability, integrity, and cycle. Results showed that sheep receiving cefazolin-loaded devices were able to avoid implant-associated infections, with normal tissue healing process. The antibiotic release followed a local concentric pattern as demonstrated by high-performance liquid chromatography detection in tissues. The in vitro results indicate the lack of relevant cytotoxic effects for the maximum antibiotic concentration released by the device. These results demonstrate the efficiency and safety of cefazolin-eluting implants in an ovine model to prevent early postsurgical infections of orthopedic devices.